EP0839798B1

EP0839798B1 - Intermediates for preparating non-peptide retroviral protease inhibitors

Info

Publication number: EP0839798B1
Application number: EP97119700A
Authority: EP
Inventors: Dale J. Kempf; Daniel W. Norbeck; John W. Erickson; Hing Leung Sham; Lynn M. Codacovi; Jacob J. Plattner
Original assignee: Abbott Laboratories
Current assignee: Abbott Laboratories
Priority date: 1989-05-23
Filing date: 1990-05-17
Publication date: 2005-08-17
Anticipated expiration: 2010-05-17
Also published as: KR0184866B1; DE69032493D1; US5670675A; KR900018134A; AU5571190A; US5545750A; ATE302180T1; HK1012337A1; HK1021530A1; IL94444A; US5142056A; IL94444A0; DE69032493T2; ES2119737T3; DK0402646T3; EP0839798A2; JPH03128335A; AU645493B2; DE69034202D1; DE69034202T2

Abstract

A retroviral protease inhibiting compound of the formula A - X - B or a pharmaceutically acceptable salt, prodrug or ester thereof, wherein X is a linking group; A is (1) substituted amino, (2) substituted carbonyl, (3) functionalized imino, (4) functionalized alkyl, (5) functionalized acyl, (6) functionalized heterocyclic or (7) functionalized (heterocyclic)alkyl; and B is (1) substituted carbonyl independently defined as herein, (2) substituted amino independently defined as herein, (3) functionalized imino independently defined as herein, (4) functionalized alkyl independently defined as herein, (5) functionalized acyl independently defined as herein, (6) functionalized heterocyclic independently defined as herein or (7) functionalized (heterocyclic)alkyl independently defined as herein. o

Description

Technical Field

This invention was made with Government support under contract number AI27220-01 awarded by the National Institute of Allergy and Infectious Diseases. The Government has certain rights in this invention.
The present invention relates to novel synthetic intermediates for making compounds for inhibiting retroviral proteases and in particular for inhibiting human immunodeficiency virus (HIV) protease, and for treating a retroviral infection and in particular an HIV infection.

Backgraund Art

Retroviruses are those viruses which utilize a ribonucleic acid (RNA) intermediate and a RNA-dependent deoxyribonucleic acid (DNA) polymerase, reverse transcriptase, during their life cycle. Retroviruses include, but are not limited to, the RNA viruses of the Retroviridae family, and also the DNA viruses of the Hepadnavirus and Caulimovirus families. Retroviruses cause a variety of disease states in man, animals and plants. Some of the more important retroviruses from a pathological standpoint include human immunodeficiency viruses (HIV-1 and HIV-2), which cause acquired immune deficiency syndrome (AIDS) in man, hepatitis B virus, which causes hepatitis and hepatic carcinomas in man, human T-cell lymphotrophic viruses I, II, IV and V, which cause human acute cell leukemia, and bovine and feline leukemia viruses which cause leukemia in domestic animals.
Proteases are enzymes which cleave proteins at specific peptide bonds. Many biological functions are controlled or mediated by proteases and their complementary protease inhibitors. For example, the protease renin cleaves the peptide angiotensinogen to produce the peptide angiotensin I. Angiotensin I is further cleaved by the protease angiotensin converting enzyme (ACE) to form the hypotensive peptide angiotensin II. Inhibitors of renin and ACE are known to reduce high blood pressure in vivo. An inhibitor of a retroviral protease should provide a therapeutic agent for diseases caused by the retrovirus.
The genomes of retroviruses encode a protease that is responsible for the proteolytic processing of one or more polyprotein precursors such as the pol and gag gene products. See Wellink, Arch. Virol. 98 1 (1988). Retroviral proteases most commonly process the gag precursor into core proteins, and also process the pol precursor into reverse transciptase and retroviral protease. In addition, retroviral proteases are sequence specific. See Pearl, Nature 328 482 (1987).
The correct processing of the precursor polyproteins by the retroviral protease is necessary for the assembly of infectious virions. It has been shown that in vitro mutagenesis that produces protease-defective virus leads to the production of immature core forms which lack infectivity. See Crawford, J. Virol. 53 899 (1985); Katoh, et al., Virology 145 280 (1985). Therefore, retroviral protease inhibition provides an attractive target for antiviral therapy. See Mitsuya, Nature 325 775 (1987).
Current treatments for viral diseases usually involve administration of compounds that inhibit viral DNA synthesis. Current treatments for AIDS (Dagani, Chem. Eng. News, November 23, 1987 pp. 41-49) involve administration of compounds such as 2',3'-dideoxycytidine, trisodium phosphonoformate, ammonium 21-tungsto-9-antimoniate, 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, 3'-azido-3'-deoxythymidine, and adriamycin that inhibit viral DNA synthesis; compounds such as AL-721 and polymannoacetate which may prevent HIV from penetrating the host cell; and compounds which treat the opportunistic infections caused by the immunosuppression resulting from HIV infection. None of the current AIDS treatments have proven to be totally effective in treating and/or reversing the disease. In addition, many of the compounds currently used to treat AIDS cause adverse side effects including low platelet count, renal toxicity and bone marrow cytopenia.
Inhibitors of HIV protease are disclosed by Moore, Biochem. Biophys. Res. Commun., 159 420 (1989); Billich, J. Biol. Chem., 263 1790S (1988); Richards, FEBS Lett., 247 113 (1989); Miller, Science 246 1149 (1989); Meek, Nature 343 90 (1990); McQuade, Science 247 454 (1990); Sigal, et al., EP-A-337714, published October 18, 1989; Kempf, et al., WO89/10752, published November 16, 1989; Molling, et al., EP-A-354522, published February 14, 1990; Sigal, et al., 357332, published March 7, 1990; Handa, et al., EP-A-346847, published December 20, 1989; Desolms, et al, EP-A-356223, published February 28, 1990; Schirlin, et al., EP-A-362002, published April 4, 1990; Dreyer, et al., WO90/00399, published January 25, 1990; and Hanko, et al., EP-A-361341, published April 4, 1990.
U.S. Patent No. 4,652,552 discloses methyl ketone derivatives of tetrapeptides as inhibitors of viral proteases. U.S. Patent No. 4,644,055 discloses halomethylketone derivatives of peptides as inhibitors of viral proteases.
None of the references mentioned above disclose or suggest the invention claimed herein.
The compounds (A-X-B) shown in Table 1 are disclosed in the following list of references. None of these references disclose or suggest the use of these compounds as inhibitors of retroviral protease or as antiviral agents.
1. S. Apparao, et al., Synthesis, 896 (1987).
2. A. Padwa, et al., J. Chem. Soc. Perkin Trans. I, 2639 (1988).
3. M. McKervey, et al., Tet. Let., 23 2509 (1982).
4. M. Fujiwara, et al., Chem. Abstr. 91:149446.
5. C. Piantadosi, et al., J. Med. Chem., 19 222 (1976).
6. M. Midland, et al., J. Org. Chem., 39 732 (1974).
7. R. Dybas, et al., U.S. Patent No. 4172094, published October 23, 1979.
8. A. Hosomi, et al., Chem. Pharm. Bull., 36 3736 (1988).
9. K. Taniguchi, et al., Chem. Abstr. 108:29541.
10. R. Freidlina, et al., Chem. Abstr. 57:16464.
11. T. Morikawa, Chem. Abstr. 54:19588.
12. M. Sorokin, et al., Chem. Abstr. 108:111518.
13. G. Ostroumova, Chem. Abstr. 72:56187.
14. T. Kamijo, et al., Chem. Pharm. Bull., 31 4189 (1983).
15. T. Takeda, et al., Bull. Chem. Soc. Jpn., 57 1863 (1984).
16. S. Kukalenko, et al., Chem. Abstr. 78:71621.
17. K. Ito, et al., Chem. Pharm. Bull., 27 1691 (1979).
18. H. Kleiner, Chem. Abstr. 73:45598.
19. G. Rawson, et al., Tetrahedron, 26 5653 (1970).
20. M. Il'ina, et al., Chém. Abstr. 70:37885.
21. E. Regel, et al., U.S. Patent No. 4,618619, issued October 21, 1986.
22. V. Plakhov, et al., Chem. Abstr. 73:30312.
23. K. Dathe, et al., Chem. Abstr. 84:73203.
24. W. Wegener, et al., Chem. Abstr. 77:101763.
25. E. Mukhametzyanova, et al., Chem. Abstr. 71:39096.
26. K. Petrov, et al., Chem. Abstr. 74:141977.
27. H. Gilman, et al., Chem. Abstr. 89:172760.
28. T. Hosokawa, et al., Bull. Chem. Soc. Jpn., 58 194 (1985).
29. G. Olah, et al., Synthesis, 221 (1980).

Disclosure of the Invention

In accordance with the present invention, there are compounds of the formula:
or a hydrochloride salt thereof or an N-protected derivative thereof.
The chiral centers of the compounds of the invention may be racemic or asymmetric. Racemic mixtures, mixtures of diastereomers, as well as single diastereomers of the compounds of the invention are included in the present invention. The terms "S" and "R" configuration are as defined by the IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, Pure Appl. Chem. (1976) 45, 13 - 30.
The terms "Ala", "Asn", "Gly", "Ile", "Leu", "Lys", "Phe", "Pro", "Ser", and "Val" as used herein refer to alanine, asparagine, glycine, isoleucine, leucine, lysine, phenylalanine, proline, serine and valine, respectively. In general, the amino acid abbreviations used herein follow the IUPAC-IUB Joint Commission on Biochemical Nomenclature for amino acids and peptides (Eur. J. Biochem. 1984, 158, 9-31).
The term "N-protecting group" or "N-protected" as used herein refers to those groups intended to protect nitrogen atoms against undesirable reactions during synthetic procedures or to prevent the attack of exopeptidases on the final compounds or to increase the solubility of the final compounds and includes but is not limited to acyl, acetyl, pivaloyl, t-butylacetyl, t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or benzoyl groups or an L- or D-aminoacyl residue, which may itself be N-protected similarly.
The term "loweralkyl" as used herein refers to straight or branched chain alkyl radicals containing from 1 to 6 carbon atoms including, but not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, n-pentyl, 1-methylbutyl, 2,2-dimethylbutyl, 2-methylpentyl, 2,2-dimethylpropyl, n-hexyl and the like.
Compounds of the invention, which are useful as intermediates, may be selected from 2-(N-benzylamino)-5-(t-butyloxycarbonylamino)-3,4-dihydroxy-1,6-diphenylhexane;
2-(N-benzyl-N-(benzyloxycarbonyl)amino)-5-(t-butyloxycarbonylamino)-3,4-dihydroxy-1,6-diphenylhexane;
5-amino-2-(N-benzylamino)-3,4-dihydroxy-1,6-diphenylhexane dihydrochloride;
2-amino-5-(t-butyloxycarbonylamino)-3,4-dihydroxy-1,6-diphenylhexane;
2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;
(2S,3R,4R,5S)-2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;
(2S,3S,4S,5S)-2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;
(2S,3R,4S,5S)-2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;
(2S,3R,4R,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane or a hydrochloride salt thereof;
(2S,3S,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane or a hydrochloride salt thereof;
(2S,3R,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane or a hydrochloride salt thereof;
(2S,3S,4R,5S)-5-amino-2-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;
and
(2S,3R,4S,5S)-2,5-di-(N-((isopropylaminocarbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.
According to preferred embodiments, such compounds may be selected from (2S,3R,4R,5S)-2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;
(2S,3S,4S,5S)-2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane; and
(2S,3R,4S,5S)-2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.
Also preferred are those compounds selected from (2S,3R,4R,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane;
(2S,3S,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane; and
(2S,3R,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane;
or a hydrochloride salt thereof.
Still further compounds of the invention are selected from (2S,3R,4S,5S)-2,5-di-(N-(Cbz-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;
(2S,3S,4S,5S)-2,5-di-(N-(Cbz-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;
(2S,3R,4R,5S)-2,5-di-(N-(Cbz-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;
(2S,3S,4S,5S)-2,5-di-(N-(valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;
(2S,3R,4S,5S)-2,5-di-(N-(valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;
(2S,3R,4R,5S)-2,5-di-(N-(valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane; and
(2S,3S,4R,5S)-2-(N-(t-butyloxy)carbonyl)amino)-5-(N-(Cbz-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.
Other intermediates of the invention are selected from 2-(N-benzyl-N-(benzyloxycarbonyl)amino)-5-(t-butyloxycarbonylamino)-1,6-diphenyl-3-hexene-3,4-oxide;
2-amino-5-(t-butyloxycarbonylamino)-1,6-diphenyl-3-hexene-3,4-oxide; and
2,5-di-(t-butyloxycarbonylamino)-1,6-diphenyl-3-hexene-3,4-oxide.
The term "alkylamino" as used herein refers to a loweralkyl appended to an NH radical.
The term "cycloalkyl" as used herein refers to an aliphatic ring having 3 to 7 carbon atoms including, but not limited to, cyclopropyl, cyclopentyl, cyclohexyl and the like. Cycloalkyl groups can be unsubstituted or substituted with one, two or three substituents independently selected from loweralkyl, haloalkyl, alkoxy, thioalkoxy, amino, alkylamino, dialkylamino, hydroxy, halo, mercapto, nitro, carboxaldehyde, carboxy, carboalkoxy and carboxamide.
The terms "alkoxy" and "thioalkoxy" as used herein refer to R₁₈O- and R₁₈S-, respectively, wherein R₁₈ is a loweralkyl group or benzyl.
The term "polyalkoxy" as used herein refers to -OR₄₅ wherein R₄₅ is a straight or branched chain containing 1-5, C_n'-O-C_n" linkages wherein n' and n" are independently selected from 1 to 3, including but not limited to methoxyethoxymethoxy, methoxymethoxy and the like.
The term "halo" or "halogen" as used herein refers to -Cl, -Br, -I or -F.
The term "haloalkyl" as used herein refers to a loweralkyl radical in which one or more of the hydrogen atoms are replaced by halogen including, but not limited to, chloromethyl, trifluoromethyl, 1-chloro-2-fluoroethyl and the like.
The term "heterocyclic ring" or "heterocyclic" as used herein refers to any 3- or 4-membered ring containing a heteroatom selected from oxygen, nitrogen and sulfur; or a 5- or 6-membered ring containing one, two or three nitrogen atoms; one nitrogen and one sulfur atom; or one nitrogen and one oxygen atom. The 5-membered ring has 0-2 double bonds and the 6-membered ring has 0-3 double bonds. The nitrogen and sulfur heteroatoms can be optionally oxidized. The nitrogen heteroatoms can be optionally quaternized.
The term "heterocyclic" also includes bicyclic groups in which any of the above heterocyclic rings is fused to a benzene ring or or a cyclohexane ring or another heterocyclic ring. Heterocyclics include: pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, furyl, thienyl and benzothienyl.
Heterocyclics also include:
Heterocyclics can be unsubstituted or monosubstituted or disubstituted with substituents independently selected from hydroxy, halo, oxo (=O), alkylimino (R*N= wherein R* is a loweralkyl group), amino, alkylamino, dialkylamino, alkoxy, polyalkoxy, haloalkyl, cycloalkyl, -COOH, -SO₃H and loweralkyl. In addition, nitrogen containing heterocycles can be N-protected.
In the compounds disclosed herein, the A, X and B components may have asymmetric centers and occur as racemates, racemic mixtures, mixtures of diastereomers and as individual diastereomers, with all isomeric forms being included.
Representative compounds which can be made using intermediates of the invention include those represented in Table 2. (In the table "Ph" represents phenyl).
The intermediates of the invention or compounds made from these intermediates can be prepared as shown in Schemes 1-5. The syntheses of segments (II) are described in the Examples. The process shown in Scheme 1 discloses the condensation of carboxylic or sulfonic acid G - OH to segments (II) in the presence of a coupling reagent to give (VI).
Coupling reagents known in the art which can be used include, but are not limited to, dicyclohexylcarbodiimide (DCC), 3-ethyl-3'-(dimethylamino)propylcarbodiimide (EDC), bis(2-oxo-3-oxazolidinyl)-phosphinic chloride (BOP-Cl), dipenylphosphoryl azide (DPPA) and the like.
In addition to the use of the carboxylic acids or sulfonic acids shown in the scheme, acid halide and other activated ester derivatives of the carboxylic acid or sulfonic acid are useful for the coupling reactions with (II). Acid halide derivatives include the acid chloride. Activated ester derivatives include activated esters commonly used by those skilled in the art for activating carboxylic acid groups for coupling with an amine to form an amide bond or for coupling with an alcohol to form an ester bond including, but not limited to, formic and acetic acid derived anhydrides, anhydrides derived from alkoxycarbonyl halides such as isobutyloxycarbonylchloride and the like, N-hydroxysuccinimide derived esters, N-hydroxyphthalimide derived esters, N-hydroxybenzotriazole derived esters, N-hydroxy-5-norbornene-2,3-dicarboxamide derived esters, 2,4,5-trichlorophenol derived esters and the like.
Compounds of the invention wherein X is -CH(OH)-CH(OH)- and R₃ is phenyl can be prepared as shown in Scheme 2. The process shown in Scheme 2 discloses the oxidation of XXVI to protected aminoaldehyde XXVII. Reductive dimerization of XXVII provides the doubly protected diaminodiol XXVIII, which is deprotected to give diaminodiol XXIX. Coupling of XXIX according to Scheme 1 provides compounds of the invention.
Compounds of the invention of the the type XXXII may be prepared as outlined in Scheme 3. Thus the known aziridine XXX (Y. L. Merrer, et al Heterocycles, 1987, 25, 541-548) is acylated (e.g. with G=Cbz-valine), sulfonated (e.g. with G=p-toluene sulfonic acid), or phosphorylated (e.g. with G=diphenylphosphinic acid), to provide compounds of the formula XXXI. These compounds are in turn treated with various nucleophiles, such as thiols, alcohols, amines, or organometallic reagents, which serve to open the aziridine ring. The acetonide can be removed by acid hydrolysis. When G is a sulfonic acid residue, e.g. p-toluenesulfonyl, this group may be removed reductively, for example, with sodium in liquid ammonia or with sodium napthalenide, to provide compounds of the formula XXXII wherein G is hydrogen. These compounds, in turn, can be N-acylated with protected amino acids, e.g. Cbz-valine, to provide additional compounds of the formula XXXII.
As outlined in Scheme 4, compounds of the invention of the formula XXXVI and XXXVII where R₃ is benzyl may be prepared by acylation of XXXIII with bromoacetyl bromide, followed by displacement of the bromide with various amine nucleophiles. R₈₀₀ and R₈₀₁ may be selected independently from hydrogen and alkyl, or may constitute a heterocyclic ring incorporating the nitrogen to which they are appended, e.g., rings such as morpholine, piperidine, and piperazine. Compounds of formula XL and XLI may be prepared by acylation of XXXIII with (chloromethyl)benzoyl chloride, followed by displacement of the benzylic chloride group with various amine nucleophiles.
As outlined in Scheme 5, the compound of the formula LXIII may be oxidized with the Swem reagent in a solvent like THF, and without isolation, treated with an organometallic reagent, such as a Grignard reagent, to provide compounds of the formula LXVII where R₃ is benzyl. These compounds, may in turn, be converted to the carbamates LXVIII with an isocyanate, wherein R₈₀₄ is hydrogen or benzyl or substituted benzyl. These carbamates may then be treated with a strong base such as potassium t-butoxide or sodium hydride in a suitable solvent such as THF, and the resulting cyclic carbamates treated with hydrogen over a palladium catalyst to provide compounds of the formula LXIX.

Example 1 (reference example)

A. 3-Hydroxy-5-phenyl-1-pentene.

Vinylmagnesium Bromide (120 mmol, 1 M) in ether was added to 80 ml of dry tetrahydrofuran and cooled under inert atmosphere to 0°C. Hydrocinnamaldehyde (8.0 ml, 61 mmol) was added dropwise, and the solution was stirred for 10 min, quenched cautiously with saturated aqueous ammonium chloride, extracted with ether, washed with saturated brine, dried over MgSO₄, and concentrated to give 9.82 g (98.6%) of the crude desired product.

B. 3-(t-Butyldimethylsilyloxy)-5-phenyl-1-pentene.

A solution of the resultant compound of Example 1A (9.82 g, 60.6 mmol) and 8.2 g (120 mmol) of imidazole in 30 ml of dimethylformamide was treated with cooling (cold water bath) with 10 g (66 mmol) of t-butyldimethylsilyl chloride and stirred at ambient temperature. After 1 h, the solution was diluted with 1:1 ether:hexane, washed with three portions of water, dried over MgSO₄, and concentrated in vacuo. Flash chromatography using 3% ethyl acetate in hexane gave 13.2 g (79%) of the desired compound as a colorless oil. ¹H NMR (CDCl₃) δ 0.04 (s, 3 H), 0.07 (s, 3 H), 0.90 (s, 9 H), 1.81 (m, 2 H), 2.66 (m, 2 H), 4.16 (q, J = 6 Hz, 1 H), 5.07 (dt, J = 10, 1 Hz, 1 H), 5.18 (dt, J = 17, 1 Hz, 1 H), 5.84 (ddd, J = 17, 10, 6 Hz, 1 H), 7.1-7.3 (m, 5 H). Mass spectrum: (M+H)⁺ = 277.

C. 2-(t-Butyldimethylsilyloxy)-4-phenylbutyraldehyde

A solution of 0.81 g (2.93 mmol) of the resultant compound of Example 1B in 20 ml of dichloromethane and 10 ml of methanol was cooled to -78°C. A mixture of ozone in air was bubbled through the solution until a blue color persisted. Air was bubbled through the solution to discharge excess ozone, and the solution was treated with dimethylsulfide. After being stirred overnight at ambient temperature, the solution was diluted with dichloromethane, washed with water, dried over MgSO₄, and concentrated in vacuo to give 0.81 g (100%) of the crude desired product.

D. (Z)-Methyl 4-(t-Butyldimethylsilyloxy)-6-phenyl-2-hexenoate.

A suspension of 120 mg (3.0 mmol) of sodium hydride (60% suspension in oil) in 20 ml of tetrahydrofuran was cooled to 0°C and treated with a solution of 937 mg (2.95 mmol) of bis(2,2,2-trifluoroethyl)(methoxycarbonylmethyl)phosphonate in 5 ml of tetrahydrofuran. The resulting solution was stirred for 10 min at 0°C, treated with a solution of 2.93 mmol of the resultant compound of Example 1C in 5 ml of tetrahydrofuran, and stirred at ambient temperature for 1 h. The solution was subsequently quenched with aqueous ammonium chloride, extracted with ether, washed with saturated brine, dried over MgSO₄, and concentrated in vacuo. Flash chromatography using 3% ethyl acetate in hexane gave 578 mg (59%) of the desired compound. ¹H NMR (CDCl₃) δ 0.02 (s, 3 H), 0.08 (s, 3 H), 0.90 (s, 9 H), 1.83 (m, 2 H), 2.64, (ddd, J = 13, 11, 6 Hz, 1 H), 2.77 (ddd, J = 13, 11, 6 Hz, 1 H), 3.70 (s, 3 H), 5.38 (br q, J = 7 Hz, 1 H), 5.72 (dd, J = 11, 1 Hz, 1 H), 6.21 (dd, J = 11, 8 Hz, 1 H), 7.15-7.3 (m, 5 H). Mass spectrum (M+H)⁺ = 335.

E . (Z)-4-(t-Butyldimethylsilyloxy)-6-phenyl-2-hexenoic Acid.

A solution of 567 mg (1.69 mmol) of the resultant compound of Example 1D in 13 ml of dioxane was cooled to 0°C, treated with 6.5 ml (3.2 mmol) of 0.5 M aqueous lithium hydroxide, and stirred at ambient temperature for 24 h. The resulting solution was poured into chloroform/1 N HCl, separated, dried over MgSO₄, and concentrated to give the desired product. ¹H NMR (CDCl₃) δ 0.04 (s, 3 H), 0.09 (s, 3 H), 0.91 (s, 9 H), 1.88 (m, 2 H), 2.66, (m, 1 H), 2.78 (m, 1 H), 5.31 (br q, J = 7 Hz, 1 H), 5.78 (dd, J = 12, 1 Hz, 1 H), 6.34 (dd, J = 12, 8 Hz, 1 H), 7.15-7.3 (m, 5 H). Mass spectrum (M+H)⁺ = 321.

F. (Z)-N-(3-Methylbutyl)-4-(t-butyldimethylsilyloxy)-6-phenyl-2-hexenamide.

A solution of the resultant compound of Example 1E (225 mg, 0.70 mmol) and 0.85 ml of 4-methylmorpholine in 15 ml of dichloromethane was cooled to 0°C and treated with 0.10 ml (0.77 mmol) of isobutyl chloroformate. The resulting solution was stirred for 10 min, treated with 0.089 ml (0.77 mmol) of isoamylamine, and stirred at ambient temperature for 2 h. The solution was subsequently diluted with dichloromethane, washed sequentially with 10% aqueous citric acid and aqueous NaHCO₃, dried over Na₂SO₄, and concentrated. Flash chromatography using 15% ethyl acetate in hexane gave 245 mg (90%) of the desired compound as an oil. ¹H NMR (CDCl₃) δ 0.04 (s, 3 H), 0.09 (s, 3 H), 0.90 (s, 9 H), 0.92 (d, J = 7 Hz, 6 H), 1.41 (br q, J = 7 Hz, 2 H), 1.63 (heptet, J = 7 Hz, 1 H), 1.86 (m, 2 H), 2.63 (m, 1 H), 2.78 (m, 1 H), 3.30 (m, 2 H), 5.50 (br q, 1 H), 5.59 (dd, J = 11, 1 Hz, 1 H), 5.98 (dd, J = 11, 8 Hz, 1 H), 7.15-7.3 (m, 5 H). Mass spectrum (M+H)⁺ = 391.

Example 2 (reference example)

A. 2-(t-Butyloxycarbonylamino)-1,5-diphenylpent-3-ene.

A solution of 15.1 g (54.5 mmol) of the resultant compound of Example 1A and 38 ml (220 mmol) of diisopropylethylamine in 450 ml of dry dichloromethane was cooled under N₂ atmosphere in an acetone/ice bath and treated dropwise with 8.5 ml (110 mmol) of methanesulfonyl chloride. The solution was stirred for 7 min after addition was complete, then was quenched with 400 ml of 10% citric acid. The bath was removed, and the mixture was extracted with 800 ml of ether. The organic layer was washed sequentially with 500 ml of water and 300 ml of saturated brine, dried over MgSO₄, and concentrated in vacuo to give the crude mesylate as an off-white solid. To a flame-dried 3-neck 1000 mL. flask equipped with an internal low-temperature thermometer was added 1.45 g (16 mmol) of anhydrous cuprous cyanide. The flask was then charged with 500 ml of anhydrous tetrahydrofuran. The suspension was cooled under N₂ altmosphere in a dry ice/acetone bath. A solution of phenylmagnesium bromide (55 ml, 165 mmol) in ether (3M) was added via syringe. The bath was removed, and the resulting beige suspension was warmed with stirring by use of a water bath. As the internal temperature reached -5°C, the solid began to dissolve, and the solution began to turn darker. By the time the internal temperature reached -1°C, the solution was homogenous, and was immediately recooled by placement of the flask in a dry ice/acetone bath. As the internal temperature reached -65°C, addition of a solution of the above crude mesylate in 75 ml of tetrahydrofuran was added via cannula. The resulting solution was stirred at ca. -70°C for 15 min. The bath was then removed, and the solution was immediately treated with 100 ml of saturated aqueous ammonium chloride followed by 300 ml of ether. As the mixture warmed, 100 ml of 1 N NH₄OH was added, and the mixture was stirred under air atmosphere for several hours while the aqueous layer turned dark blue. The mixture was then extracted with 500 ml of ether. The organic layer was washed with saturated brine and concentrated in vacuo without drying to give a yellow oil. The combined aqueous layers were extracted with 500 ml of additional ether, which was added to the above oil. The resulting solution was washed with saturated brine, dried over MgSO₄, and concentrated to a yellow oil. The oil was taken up in 100 ml of dichloromethane, treated with 50 g of silica gel, and concentrated in vacuo until the residue was a freely flowing solid. The solid was placed on top of a 60 mm column containing 300 g of silica gel and eluted sequentially with 1200 ml of hexane (to bring out biphenyl formed as a side product) followed by 5000 ml of 5% ethyl acetate in hexane. Combination of the pure fractions gave 11.95 g (65%) of the desired compound. ¹H NMR (CDCl₃, major isomer) δ 1.40 (s, 9 H), 2.7-2.9 (m, 2 H), 3.32 (d, J = 7 Hz, 2 H), 4.4 (br, 2 H), 5.43 (dd, J = 15, 6 Hz, 1 H), 5.64 (dt, J = 15, 7 Hz, 1 H), 7.0-7.3 (m, 10 H).

B. 2-(t-Butyloxycarbonylamino)-1,5-diphenylpent-3-ene-3,4-oxide.

A solution of 11.71 g (34.75 mmol) of the resultant compound of Example 2A in 200 ml of dichloromethane was treated with 15 g (174 mmol) of solid sodium bicarbonate, cooled to 0°C, and treated with 24 g (69 mmol) of m-chloroperbenzoic acid (50%). The resulting suspension was sealed with a septum and stirred in a cold room (5°C) for three days. The resulting mixture, which contained much precipitate, was decanted into a 1000 ml flask. The white residue was broken up and washed out with 400 ml of 10% sodium thiosulfate solution and 300 ml of ether. The two-phase mixture was stirred for 2 hours, and the layers were separated. The organic layer was washed sequentially with 200 ml portions of 2 M NaOH, water, and saturated brine. The combined aqueous layers were extracted with 200 ml of ether, which was washed sequentially with 50 ml of water and 50 mL of aqueous brine, combined with the original organic phase, dried over MgSO₄, and concentrated in vacuo. The resulting oil was taken up in 100 ml of dichloromethane, treated with 50 g of silica gel, and concentrated in vacuo until the residue was a freely flowing solid. The solid was placed on top of a 60 mm column containing 300 g of silica gel and eluted sequentially with 1000 ml of 5% ethyl acetate in hexane followed by 3500 ml of 12% ethyl acetate in hexane. Concentration of the combined fractions gave 9.36 g (76%) of the desired compound (ca. 4:1 mixture of diastereomers) as an oil which solidified upon standing.

C. 4-Azido-2-(t-butyloxycarbonylamino)-1,5-diphenyl-3-hydroxypentane.

A solution of 9.12g (25.84 mmol) of the resultant compound of Example 2B 7.0 g (140 mmol) of lithium azide, and 1.73 g (32 mmol) of ammonium chloride in 75 ml of dimethylformamide and 7.5 ml of water was heated in an oil bath at 70°C for 32 hours. After being allowed to cool, the resulting solution was treated with 1000 ml of 1:1 ether/hexane and 800 ml of water. The layers were separated, and the aqueous layer was extracted with 500 ml of additional 1:1 ether/hexane. The combined organic layers were washed sequentially with 400 ml of water and 200 ml of saturated brine, dried over MgSO₄, and concentrated in vacuo to a solid. The solid was taken up in 100 ml of dichloromethane, treated with 50 g of silica gel, and concentrated in vacuo until the residue was a freely flowing solid. The solid was placed on top of a 60 mm column containing 300 g of silica gel and eluted sequentially with 1000 ml of 10% ethyl acetate in hexane, 1000 ml of 15% ethyl acetate in hexane, and 2000 ml of 25% ethyl acetate in hexane. Concentration of the fractions gave 9.26 g (91%) of the desired compound as a ca. 4:1 mixture of diastereomers. ¹H NMR (CDCl₃) δ 1.42 (s, 9 H), 2.78 (m, 1 H), 2.89 (m, 1 H), 3.13 (m, 1 H), 3.29 (m, 1 H), 3.41 (m, 1 H), 3.53 (m, 1 H), 3.80 (m, 1 H), 4.06 (m, 1 H), 4.83 (m, 1 H), 7.2-7.35 (m, 10 H). Mass spectrum (M+H)⁺ = 338.

Example 3 (reference example)

4-Amino-2-(t-butyloxycarbonylamino)-1,5-diphenyl-3-hydroxypentane.

A rapidly stirring suspension of 10 mg of 10% palladium on carbon in 0.3 ml of methanol was treated under inert atmosphere with 60 mg (0.95 mmol) of solid ammonium formate. After 3 min, a solution of 52 mg (0.13 mmol) of the resultant compound of Example 2C in 0.4 ml of methanol was added. The resulting mixture was stirred for 2 h, diluted with methanol and 1 N ammonium hydroxide, filtered through Celite, and concentrated in vacuo. The residue was treated with 1 N NaOH, extracted with two portions of chloroform, dried over sodium sulfate, and concentrated. Flash chromatography using 7.5% methanol in chloroform gave 37 mg (76%) of the desired compound (R_f 0.38, 2.5% methanol/2% isopropylamine in chloroform) as a white solid, m.p. 134-135°C. ¹H NMR (CDCl₃) δ 1.48 (s, 9 H), 2.50 (dd, J = 13, 10 Hz, 1 H), 2.8-3.1 (m, 4 H), 3.41 (br d, J = 7 Hz, 1 H), 4.11 (br q, J = 8 Hz, 1 H), 4.83 (br d, J = 9 Hz, 1 H), 7.15-7.35 (m, 10 H). Mass spectrum (M+H)⁺ = 370.
Anal. Calcd. for C₂₂H₃₀N₂O₃·0.15H₂O: C, 70.81; H, 8.18; N, 7.51. Found: C, 70.89; H, 8.15; N, 7.43.

Example 4

A. trans-2-(N-Benzyl-N-(benzyloxycarbonyl)amino)-5-(t-butyloxycarbonylamino)-1,6-diphenyl-3-hexene.

Using the procedure of Example 2A but replacing phenylacetaldehyde with N-benzyl-N-(benzyloxycarbonyl)-phenylalaninal gave, after flash chromatography using 20% ethyl acetate in hexane, 45 mg (30%) of the desired compound. ¹H NMR (d₆-DMSO, 100°C) δ 1.32 (s, 9 H), 2.54 (dd, J = 14, 7 Hz, 1 H), 2.62 (dd, J = 14, 7 Hz, 1 H), 2.77 (dd, J = 14, 7 Hz, 1 H), 2.83 (dd, J = 14, 7 Hz, 1 H), 4.04 (br pentet, J = 7 Hz, 1 H), 4.20 (d, J = 16 Hz, 1 H), 4.33 (d, J = 16 Hz, 1 H), 4.48 (br q, J = 7 Hz, 1 H), 5.03 (AA', 2 H), 5.44 (dd, J = 16, 6 Hz, 1 H), 5.61 (dd, J = 16, 7 Hz, 1 H), 7.0-7.4 (m, 20 H). Mass spectrum (M+H)⁺ = 591.

B. 2-(N-Benzyl-N-(benzyloxycarbonyl)amino)-5-(t-butyloxycarbonylamino)-3,4-dihydroxy-1,6-diphenyl-3-hexane.

A solution of 40 mg (0.068 mmol) of the resultant compound of Example 4A in 1 ml of tetrahydrofuran was treated sequentially with 0.034 ml (0.0034 mmol) of osmium tetroxide (2.5% in t-butanol) and 20 mg (0.14 mmol) of 4-methylmorpholine-N-oxide. After 20 h, the solution was treated with 10% Na₂S₂O₃, stirred for 15 min, diluted with ether, washed with two portions of 10% Na₂S₂O₃, one portion of water, and one portion of saturated aqueous NaHCO₃, dried over Na₂SO₄, and concentrated in vacuo. Flash chromatography using 30% ethyl acetate in hexane gave the desired compound (R_f 0.43, 30% ethyl acetate in hexane) as a 2:1 mixture of diastereomers. Mass spectrum (M+H)⁺ = 625.

Example 5

2-(N-Benzylamino)-5-(t-butyloxycarbonylamino)-3,4-dihydroxy-1,6-diphenyl-3-hexane.

Ammonia (ca. 3 ml) was condensed into a precooled (-78°C) mixture of excess sodium metal in 2 ml of tetrahydrofuran. A solution of 25 mg (0.040 mmol) of the resultant compound of Example 4B in 1 ml of tetrahydrofuran was added, and the resulting solution was stirred for 10 min, quenched with saturated aqueous ammonium chloride, allowed to warm to ambient temperature, extracted with ether, dried over Na₂SO₄, and concentrated to give the crude desired product. Mass spectrum: (M+H)⁺ = 491.

Example 6

5-Amino-2-(N-benzylamino)-3,4-dihydroxy-1,6-diphenyl-3-hexane Dihydrochloride.

The resultant compound of Example 5 (18.5 mg, 0.038 mmol) was treated with 1 ml of 4 M HCl in dioxane. After 1 h, the solution was concentrated in vacuo to give the desired compound.

Example 7 (reference example)

A. (Z)-(4-(t-Butyldimethylsilyloxy)-6-phenyl-2-hexenoyl)-Val-Val Amide.

A solution of the resultant compound of Example 1E (57 mg, 0.18 mmol) and 0.022 ml of 4-methylmorpholine in 3 ml of dichloromethane was cooled to 0°C and treated with 0.026 ml (0.19 mmol) of isobutyl chloroformate. The resulting solution was stirred for 10 min, treated with a solution of 41 mg (0.19 mmol) of H-Val-Val-NH₂ in 1.5 ml of dimethylformamide, and stirred at ambient temperature for 2 h. The solution was subsequently diluted with ethyl acetate, washed sequentially with 10 % aqueous citric acid and aqueous NaHCO₃, dried over MgSO₄, and concentrated. Flash chromatography using 60% ethyl acetate in chloroform gave 53 mg (57%) of the desired compound as a 1:1 mixture of diastereomers.

B. (Z)-(4-Hydroxy-6-phenyl-2-hexenoyl)-Val-Val Amide.

A solution of 13 mg (0.025 mmol) of the resultant compound of Example 7A in 0.5 ml of tetrahydrofuran was treated with 0.065 ml (0.065 mmol) of tetra-n-butylammonium fluoride (1 M in tetrahydrofuran). After being stirred for 16 h, the solution was concentrated in vacuo. Flash chromatography using 7.5% methanol in chloroform gave the desired compound, m.p. 147-149°C, as a 1:1 mixture of diastereomers (R_f 0.15, 7.5% methanol in chloroform). Mass spectrum: (M+H)⁺ = 403.
Anal. Calcd. for C₂₂H₃₃N₃O₄·0.5H₂O: C, 64.05; H, 8.31; N, 10.19. Found: C, 63,84; H, 7.65; N, 10.06.

Example 8 (reference example)

2-(t-Butyloxycarbonylamino)-4-(Cbz-valinyl-amino)-1,5-diphenyl-3-hydroxypentane.

A solution of 39 mg (0.16 mmol) of Cbz-Val-OH, 52 mg (0.14 mmol) of the resultant compound of Example 3 and 23 mg (0.17 mmol) of 1-hydroxybenzotriazole in 2 ml of dimethylformamide was treated with 0.019 ml (0.17 mmol) of 4-methylmorpholine, cooled to 0°C, and treated with 33 mg (0.17 mmol) of N-ethyl-N'-(dimethylaminopropyl)-carbodiimide hydrochloride. After being stirred at ambient temperature overnight, the solution was diluted with ethyl acetate, washed sequentially with 10% citric acid, water, and aqueous NaHCO₃, dried over Na₂SO₄, and concentrated in vacuo. Separation of the desired compounds by flash chromatography using methanol in chloroform gave 80 mg (95%) of the desired compound (R_f 0.40, 10% methanol in chloroform) as a white solid, m.p. 187-187.5°C. Mass spectrum (M+H)⁺ = 604.

Example 9 (reference example)

A. 2-Amino-4-azido-1,5-diphenyl-3-hydroxypentane Hydrochloride.

The resultant compound of Example 2C (25 mg, 0.063 mmol) was treated with 1 ml of 4 M HCl in dioxane, stirred for 0.5 h at ambient temperature, and concentrated in vacuo to give the desired compound.

Example 10

5-((N-Acetyl-valinyl-valinyl)amino)-2-(N-benzylamino)-3,4-dihydroxy-1,6-diphenyl-3-hexane.

A solution of 10 mg (0.04 mmol) of Ac-Val-Val-OH, 0.039 mmol of the resultant compound of Example 6, and 6 mg (0.044 mmol) of 1-hydroxybenzotriazole in 1 ml of dichloromethane and 0.4 ml of dimethylformamide was treated with 0.009 ml (0.08 mmol) of 4-methylmorpholine, cooled to 0°C, and treated with 9 mg (0.047 mmol) of N-ethyl-N'-(dimethylaminoethyl)carbodiimide. After being stirred at ambient temperature overnight, the solution was diluted with ethyl acetate, washed sequentially with aqueous NaHCO₃ and water, dried over Na₂SO₄, and concentrated in vacuo. Separation of the desired compounds by flash chromatography using 6% methanol in chloroform gave 3.0 mg (13%) of the less.polar diastereomer (R_f 0.33, 10% methanol in chloroform), m.p. 154-156°C, and 4.1 mg (17%) of the more polar diastereomer (R_f 0.28), m.p. 121-124°C. Mass spectrum (for each diastereomer): (M+H)⁺ = 631.

Example 11 (reference example)

2-Amino-4-(Cbz-valinyl-amino)-1,5-diphenyl-3-hydroxypentane.

Using the procedure of Example 9 with the resultant compound of Example 8 gave, after silica gel chromatography using methanol/ isopropylamine/ chloroform, the desired compound (R_F 0.3; 2.5% methanol/ 2% isopropylamine/ chloroform) in 100% yield, m.p. 158-160°C. Mass spectrum (M+H)⁺ = 504.

Example 12 (reference example)

2,4-Bis-(Cbz-valinyl-amino)-1,5-diphenyl-3-hydroxypentane.

Using the procedure of Example 8 but replacing the resultant compound of Example 3 with the resultant compound of Example 11 gave, after silica gel chromatography using methanol/ chloroform, the desired compound (R_F 0.4; 2.5% methanol/ 2% isopropylamine/ chloroform) in 98% yield, m.p. 198-200°C. Mass spectrum (M+H)⁺ = 737.
Anal. Calcd. for C₄₃H₅₂N₄O₇·0.5H₂O: C, 69.24; H, 7.16; N, 7.51. Found: C, 69.40; H, 7.29; N, 7.47.

Example 13 (reference example)

A. N-Boc-valinyl-valine benzyl ester.

Boc-Val-OH (2.86 g, 13.2 mmol) was coupled to valine benzyl ester p-toluenesulfonate (5.0 g, 13.2 mmol) using the procedure of Example 8 to give 5.41g, (100%) of the desired product (R_F 0.15; 20% ethyl acetate in hexane) as a colorless gum. Mass spectrum (M+H)⁺ = 407.

B. N-(5-Carbomethoxypentanoyl)-valinyl-valine benzyl ester.

The resultant compound of Example 13A (0.50 g, 1.23 mmol) was deprotected according to the procedure of Example 9 and coupled to adipic acid monomethyl ester (0.21 g, 1.28 mmol) using the mixed anhydride procedure of Example 7A to give, after flash chromatography using 40% ethyl acetate in chloroform, 0.53 g (96%) of the desired compound.

C. N-(5-Carbomethoxypentanoyl)-valinyl-valine.

A mixture of the resultant compound of Example 13B (0.53 g, 1.18 mmol) and 100 mg of 10% palladium on carbon in 30 ml of methanol was stirred under one atmosphere of hydrogen. After 5 h, the mixture was filtered through Celite and concentrated to give 0.40 g (93%) of the desired compound as a solid.

Example 14 (reference example).

A. (4S)-3-(4-Methylpentanoyl)-4-(2-propyl)oxazolidine-2-one.

To a stirred solution of 4-(2-propyl)-oxazolidine-2-one in anhydrous tetrahydrofuran (250 ml) under a nitrogen atmosphere at -78°C was added in a dropwise fashion a solution of n-butyllithium in hexane (50 ml, 77.4 mmol) over 5 to 10 min. After stirring an additional 20 min at -78°C, 4-methylpentanoyl chloride (85.2 mmol) was added neat. The reaction was warmed to room temperature and stirred 1 to 2 h at the temperature. The reaction was quenched by adding 100 ml of saturated aqueous ammonium chloride and the volatiles were removed by rotary evaporation. The resulting aqueous residue was extracted three times with ether and the combined organic phases were washed with brine, dried (Na₂SO₄), filtered and concentrated in vacuo. Recrystallization from hexanes/ethyl acetate provided the desired compound.

B. (4R)-3-((2-R)-2-(t-Butyloxycarbonyl)methyl-4-methylpentanoyl)-4-(2-propyl)oxazolidine-2-one.

To a stirred solution of the resultant compound of Example 14A (8.72 mmol) in anhydrous tetrahydrofuran (30 ml) under a nitrogen atmosphere at -78°C was added a solution of sodium hexamethyldisilylamide (9.6 ml, 9.59 mmol) in tetrahydrofuran. After stirring for 30 min at -78°C, t-butyl bromoacetate (2.21 g, 11.34 mmol) was added in anhydrous tetrahydrofuran and the resulting solution stirred 1 h at -78°C. The reaction was quenched by adding 20 ml of saturated aqueous ammonium chloride and partitioned between water and ether. The aqueous layer was drawn off and extracted with ether. The combined organic phases were washed with 10% aqueous HCl, saturated aqueous NaHCO₃, and brine, dried (Na₂SO₄), filtered, and concentrated in vacuo. Recrystallization from acetone/hexanes provided the desired compound.

C. Benzyl-(2R)-2-(t-Butyloxycarbonyl)methyl-4-methylpentanoate.

To a stirred solution of dry benzyl alcohol (0.55 ml, 5.33 mmol) in anhydrous tetrahydrofuran (18 ml) under a nitrogen atmosphere at 0°C was added a hexane solution of n-butyllithium (2.58 ml; 4.00 mmol). To this solution was added the resultant compound of Example 14B in anhydrous tetrahydrofuran (10 ml). After stirring 1 h at 0°C the reaction was quenched by adding excess saturated aqueous ammonium chloride. The volatiles were removed by rotary evaporation and the resulting aqueous residue was extracted two times with ether. The combined organic layers were washed with brine, dried (Na₂SO₄), filtered, and concentrated in vacuo to provide an oil which was purified by chromatography on SiO₂ to give the desired compound.

D. Benzyl (2R)-2-(Carboxymethyl)-4-methylpentanoate.

The resultant compound of Example 14C (1.47 mmol) was dissolved in a 1:1 (v:v) solution (6 ml) of trifluoroacetic acid and dichloromethane and stirred at room temperature for 1 h. The volatiles were removed in vacuo to provide the desired compound. The unpurified material was of sufficient purity to employ in subsequent steps.

Example 15 (reference example)

A. Methyl a-Benzylacrylate.

a-Benzylacrylic acid (1.00 g, 6.17 mmol) in methanol (20 ml) was treated with BF₃·Et₂O (2 ml). The mixture was heated to reflux for 14 h, cooled, and poured into saturated NaHCO₃ solution. Extraction with ether followed by drying over Na₂SO₄ and evaporation afforded 1.03 g (95%) of a mobile oil. ¹H NMR (CDCl₃) δ 7.17-7.35 (m,5H), 6.23 (m,1H), 5.47 (m,1H), 3.74 (s,3H), 3.63 (s,2H).

B. Methyl 2-Benzyl-3-(N-methoxyl-N-methylamino)propionate.

The resultant compound from Example 15A (800 mg, 4.54 mmol), N-methyl,O-methylhydroxylamine hydrochloride (0.57 g, 5.4 mmol), and NaHCO₃ (0.46 g, 5.48 mmol) in dimethylsulfoxide (5 ml) were heated at 130°C for 20 h. The mixture was diluted with ethyl acetate, washed with water, saturated NaHCO₃ solution and brine, and then was dried over Na₂SO₃ and evaporated. Chromatography of the residue on silica gel with 10% ethyl acetate in hexane afforded 226 mg (21%) of a mobile oil. ¹H NMR (CDCl₃) δ 7.10-7.30 (m,5H), 3.60 (s,3H), 3.47 (s,3H), 2.80-3.10 (m,4H), 2.60 (dd,1H), 2.55 (s,3H).

Example 16 (reference example)

A. Methyl 2-Benzyl-3-pyrazol-1-ylpropionate.

Using the procedure of Example 15B but replacing N-methyl,O-methylhydroxylamine hydrochloride and NaHCO₃ with pyrazole provided the desired product as an oil. ¹H NMR (CDCl₃) δ 7.52 (d, 1H), 7.10-7.35 (m,6H), 6.10 (dd,1H), 4.38 (dd,1H), 4.24 (dd,1H), 3.57 (s,3H), 3.37 (m,1H), 2.98 (dd,1H), 2.82 (dd,1H).
coupled to the resultant compound of Example 138 to give the desired compound.

Example 17 (reference example)

2,4-Bis-N-(valinyl)amino-1,5-diphenyl-3-hydroxypentane.

The resultant compound of Example 12 was hydrogenolyzed according to the procedure of Example 13C to provide the desired compound (R_f 0.1, 10% methanol in chloroform) as a white solid, m.p. 131-132°C.

Example 18

2-Amino-5-(t-butyloxycarbonylamino)-3,4-dihydroxy-1,6-diphenyl-3-hexane.

A mixture of 0.13 g of the resultant compound of Example 4B and 0.13 g of 10% palladium on carbon in 50 ml of ethyl acetate was shaken under 4 atmospheres of H₂ for 4 h. The resulting mixture was filtered through Celite and concentrated in vacuo to provide 72 mg (86%) of the desired compound as a 1:1 mixture of diastereomers. Mass spectrum: (M + H)⁺ = 401.

Example 19 (reference example)

A. N,N-Bis-((2-methoxycarbonyl)-3-phenylpropyl)-O-benzylhydroxylamine.

The resultant compound from Example 15A (4 mmol), O-benzylhydroxylamine hydrochloride (2 mmol), and NaHCO₃ (2.2 mmol) in dimethylsulfoxide (5 ml) were heated at 130°C for 20 h. The mixture was diluted with ethyl acetate, washed with water, saturated NaHCO₃ solution and brine, and then was dried over Na₂SO₃ and evaporated. Silica gel chromatography provided the desired compound.

Example 20 (reference example)

A. α-Isocyanato-valine Methyl Ester.

A suspension of L-valine methyl ester hydrochloride (10 g) in toluene (400 ml) was heated to 100°C and phosgene gas was bubbled into the reaction mixture. After approximately 6 h, the mixture became homogeneous. The bubbling of phosgene was continued for 10 more min, then the solution was cooled with the bubbling of N₂ gas. The solvent was then evaporated and the residue chased with toluene two times. Evaporation of solvent gave 14.2 g of the crude desired compound.

B. N-((4-Pyridinyl)methoxycarbonyl)-valine Methyl Ester.

A solution of 0.73 g (4.65 mmol) of the resultant compound of Example 20A and 0.51 g (4.65 mmol) of pyridine-4-methanol in 30 mL of toluene was heated at reflux under N₂ atmosphere for 4 h. The solvent was removed in vacuo, and the residue was purified by silica gel chromatography using 2% methanol in chloroform to give 1.01 g (82%) of the desired compound as an oil. ¹H NMR (CDCl₃) δ 0.91 (d, J = 7 Hz, 3 H), 0.99 (d, J = 7 Hz, 3 H), 2.19 (m, 1 H), 3.76 (s, 3 H), 4.31 (dd, J = 9, 5 Hz, 1 H), 5.12 (s, 2 H), 5.37 (br d, 1 H), 7.25 (d, J = 6 Hz, 2 H), 8.60 (d, J = 6 Hz, 2 H).

C. N-((4-Pyridinyl)methoxycarbonyl)-valine Lithium Salt.

A solution of 50.8 mg (0.191 mmol) of the resultant compound of Example 20B in 0.75 ml of dioxane was treated with 0.46 ml (0.23 mmol) of 0.5 M aqueous lithium hydroxide. The resulting solution was stirred overnight at ambient temperature and concentrated in vacuo to provide the desired compound.

D. 2-(N-((4-Pyridinyl)methoxycarbonyl)-valinyl-amino)-4-(Cbz-valinyl-amino)-1,5-diphenyl-3-hydroxypentane.

A solution of the resultant compound of Example 20C (0.191 mmol), 94 mg (0.97 mmol) of the resultant compound of Example 11, and 31 mg (0.23 mmol) of 1-hydroxybenzotriazole in 2 ml of dimethylformamide was treated under N₂ atmosphere with 44 mg (0.23 mmol) of N-ethyl-N'-(dimethylaminopropyl) carbodiimide hydrochloride and stirred overnight at ambient temperature. The resulting solution was taken up in ethyl acetate, washed sequentially with aqueous NaHCO₃, H₂O, and saturated brine, dried over Na₂SO₄, and concentrated in vacuo. Chromatography on silica gel using 3% methanol in chloroform provided 119 mg (87%) of the desired compound (R_f 0.19, 5% methanol in chloroform) as a white solid, m.p. 170-172°C (dec). Mass spectrum: (M + 1)⁺ = 738.
Anal. Calcd for C₄₂H₅₁N₅O₇·0.5H₂O: C, 67.54; H, 7.02; N, 9.38. Found: C, 67.53; H, 7.00; N, 9.39.

Example 21 (reference example)

A. N-((3-Pyridinyl)methoxycarbonyl)-valine Methyl Ester.

Using the procedure of Example 20B but replacing pyridine-4-methanol with pyridine-3-methanol provided the desired compound as an oil after silica gel chromatography using 2% methanol in chloroform. ¹H NMR (CDCl₃) δ 0.90 (d, J = 7 Hz, 3 H), 0.98 (d, J = 7 Hz, 3 H), 2.16 (m, 1 H), 3.65 (s, 3 H), 4.30 (dd, J = 9, 5 Hz, 1 H), 5.14 (s, 2 H), 5.30 (br d, 1 H), 7.30 (dd, J = 8, 5 Hz, 1 H), 7.70 (br d, J = 8 Hz, 1 H), 8.58 (dd, J = 4, 1 Hz, 1 H), 8.63 (br s, 1 H).

B. N-((3-Pyridinyl)methoxycarbonyl)-valine Lithium Salt.

Using the procedure of Example 20C with the resultant compound of Example 21A provided the desired compound.

Example 22 (reference example)

A. N-((2-Pyridinyl)methoxycarbonyl)-valine Methyl Ester.

Using the procedure of Example 20B but replacing pyridine-4-methanol with pyridine-2-methanol provided 0.72 g (54%) of the desired compound as an oil after silica gel chromatography using 2% methanol in chloroform. ¹H NMR (CDCl₃) δ 0.91 (d, J = 7 Hz, 3 H), 0.98 (d, J = 7 Hz, 3 H), 2.19 (m, 1 H), 3.75 (s, 3 H), 4.32 (dd, J = 9, 5 Hz, 1 H), 5.24 (s, 2 H), 5.39 (br d, 1 H), 7.23 (ddd, J = 8, 4, 1 Hz, 1 H), 7.37 (d, J = 8 Hz, 1 H), 7.70 (td, J = 8, 2 Hz, 1 H), 8.60 (br d, 1 H).

B. N-((2-Pyridinyl)methoxycarbonyl)-valine Lithium Salt.

Using the procedure of Example 20C with the resultant compound of Example 22A provided the desired compound.

Example 23 (reference example)

A. N-((3-Pyridinyl)carbonyl)-valine Benzyl Ester.

A solution of 2.44 g (6.44 mmol) of L-valine benzyl ester p-toluenesulfonate in 100 ml of dichloromethane was cooled under N₂ atmosphere to 0°C and treated sequentially with 1.15 g (6.44 mmol) of nicotinyl chloride hydrochloride and 2.8 ml (26 mmol) of 4-methylmorpholine. After being stirred at ambient temperature overnight, the resulting solution was diluted with 200 ml of diethyl ether, washed sequentially with water, aqueous NaHCO₃, and saturated brine, dried over Na₂SO₄, and concentrated in vacuo to give 2.09 g (95%) of the desired compound. ¹H NMR (CDCl₃) δ 0.96 (d, J = 7 Hz, 3 H), 1.01 (d, J = 7 Hz, 3 H), 2.30 (m, 1 H), 4.83 (dd, J = 9, 5 Hz, 1 H), 5.20 (AA', 2 H), 6.67 (br d, 1 H), 7.37 (br s, 6 H), 8.11 (dd, J = 8, 2 Hz, 1 H), 8.74 (br, 1 H), 9.01 (s, 1 H).

B. N-((3-Pyridinyl)carbonyl)-valine.

A suspension of 0.16 g of 10% palladium on carbon in 20 ml of methanol was treated with a solution of 1.08 g (3.16 mmol) of the resultant compound of Example 23A in 10 ml of methanol. The resulting mixture was stirred vigorously under H₂ atmosphere for 4 h, filtered through Celite, and concentrated in vacuo to provide the desired compound as an off-white solid.

Example 24 (reference example)

A. N-((2-(4-Morpholinyl)ethyloxy)carbonyl)-valine Methyl Ester.

A solution of 1.04 g (6.60 mmol) of the resultant compound of Example 20A and 0.88 ml (7.25 mmol) of 4-(2-hydroxyethyl)morpholine in 30 mL of toluene was heated at reflux under N₂ atmosphere for 12 h. The solvent was removed in vacuo, and the residue was purified by silica gel chromatography using 5% methanol in chloroform to give 1.41 g (71%) of the desired compound as an oil. ¹H NMR (CDCl₃) δ 0.89 (d, J = 7 Hz, 3 H), 0.96 (d, J = 7 Hz, 3 H), 2.16 (m, 1 H), 2.50 (br t, 4 H), 2.62 (t, J = 6 Hz, 2 H), 3.72 (t, J = 6 Hz, 4 H), 3.75 (s, 3 H), 4.20 (br t, 2 H), 4.37 (dd, J = 9, 5 Hz, 1 H), 5.25 (br d, 1 H).

B. N-((2-(4-Morpholinyl)ethyloxy)carbonyl)-valine Lithium Salt.

A solution of 77.7 mg (0.27 mmol) of the resultant compound of Example 24A in 1 ml of dioxane was treated with 1.04 ml (0.52 mmol) of 0.5 M aqueous lithium hydroxide. After being stirred for 2.5 h at ambient temperature, the resulting solution was treated with 0.26 ml (0.26 mmol) of 1 N aqueous HCl and concentrated in vacuo to provide the desired compound as a white solid.

Example 25

A. N-((t-Butyloxy)carbonyl)-phenylalaninal.

A solution of 2.8 ml (40 mmol) of dry dimethylsulfoxide in 150 ml of dry dichloromethane was cooled under nitrogen atmosphere in a dry ice/chloroform bath (ca. -60°C). In a separate flask, a 2 M solution of oxalyl chloride in dichloromethane (15 ml, 30 mmol) was precooled to -60°C and then added via cannula. After 10 min, a solution of 5 g (20 mmol) of N-((t-butyloxy)carbonyl)-phenylalaninol in 30 ml of dry dichloromethane was added via cannula. The resulting solution was stirred at -60°C for 45 min, and was subsequently treated via syringe with 11 ml (80 mmol) of dry triethylamine. After being stirred for an additional 15 min at -60°C, the solution was quenched by addition of 10% aqueous citric acid, then immediately poured into a rapidly stirred mixture of 200 ml of 1:1 hexane:ether and 100 ml of 10% aqueous citric acid. The reaction flask was rinsed with ether which was added to the above mixture. The mixture was poured into a separatory funnel and the layers were separated. The aqueous layer was washed sequentially with dilute aqueous sodium bicarbonate and saturated brine, dried over MgSO₄, and concentrated in vacuo to provide the crude desired compound.

B. 2,5-Di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

In a glove bag purged with argon, a 500 ml three-neck flask was charged with 27 g of TiCl₃(DME)₂ followed by 200 ml of anhydrous dimethoxyethane (DME). A separate flask was charged with 20 g of Zn-Cu couple and connected to one of the side necks of the three-neck flask with Gooch tubing. The flask was sealed with septa, removed from the glove bag, and outfitted under positive argon flow with an overhead mechanical stirrer. Under positive argon pressure, Zn-Cu was added in portions with vigorous stirring. After addition, the Gooch tubing was removed and replaced with a rubber septum. Stirring was continued while the flask was placed in an oil bath and heated to 85°C for 2.5 h. After being allowed to cool, the flask was placed in an ice bath while stirring was continued, and the mixture was treated via cannula with a solution of the resultant compound of Example 25A (20 mmol) in 20 ml of anhydrous dimethoxyethane. The progress of the reaction was monitered by tlc. After 1 h, the reaction mixture was filtered through Celite, and the residue was washed with ethyl acetate. The filtrate was treated with saturated aqueous sodium bicarbonate, and air was bubbled through the suspension until it became white. The layers were separated, and the organic layer was washed with saturated brine, dried over MgSO₄, and concentrated to give 3.7 g of a light yellow solid. The solid was taken up in dichloromethane, treated with silica gel, and concentrated to a freely flowing powder. The powder was placed on the top of a silica gel column and eluted first with 70:30 hexane:ethyl acetate to bring off the more mobile product (R_f 0.26, 70:30 hexane:ethyl acetate) which contained two diastereomers (2S,3S,4S,5S and 2S,3R,4S,5S) followed by 60:40 hexane:ethyl acetate to bring off the less mobile product (R_f 0.10) which contained one major diastereomer (2S,3R,4R,5S).
(2S,3R,4R,5S)-2,5-Di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane: m.p. 200-202°C. ¹H NMR δ 1.35 (s, 18 H), 2.87 (dd, J = 13, 7 Hz, 2 H), 2.98 (dd, J = 13, 7 Hz, 2 H), 3.41 (m, 2 H), 3.76 (br q, J = 8 Hz, 2 H), 3.96 (m, 2 H), 4.77 (br d, J = 8 Hz, 2 H), 7.15-7.3 (m, 10 H). Mass spectrum: (M + H)⁺ = 501.
Anal. Calcd for C₂₈H₄₀N₂O₆·0.5H₂O: C, 65.99; H, 8.11; N, 5.50. Found: 65.96; H, 7.96; N, 5.49.

Example 26

(2S,3R,4R,5S)-2,5-Diamino-3,4-dihydroxy-1,6-diphenylhexane.

(2S,3R,4R,5S)-2,5-Di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane (2.7 g, 5.4 mmol) was treated with 200 ml of 6N aqueous hydrochloric acid and heated to 90°C until the solid had completely dissolved (30 min). The resulting solution was cooled, concentrated in vacuo, treated with saturated brine and 3N aqueous NaOH, extracted with chloroform, dried over Na₂SO₄, and concentrated in vacuo. Silica gel chromatography using 3% methanol/2% isopropylamine in chloroform provided the pure desired compound (R_f 0.40, 5% methanol/2% isopropylamine in chloroform) as a white solid, m.p. 86-89°C. ¹H NMR (CDCl₃) δ 2.72 (dd, J = 14, 9 Hz, 2 H), 2.92 (dd, J = 14, 6 Hz, 2 H), 3.03 (dd, J = 9, 5 Hz, 2 H), 3.69 (s, 2 H), 7.15-7.35 (m, 10 H). Mass spectrum: (M + H)⁺ = 301.
Anal. Calcd for C₁₈H₂₄N₂O₂·0.25H₂O: C, 70.91; H, 8.10; N, 9.19. Found: C, 70.52; H, 7.92; N, 8.93.

Example 27

A. N-((Cbz-valinyl)oxy)-succinimide

A suspension of 3.40 g (13.5 mmol) of Cbz-valine and 1.56 g (13.5 mmol) of N-hydroxysuccinimide in 200 ml of dichloromethane was treated with 2.86 g (14.9 mmol) of N-ethyl-N'-(dimethylaminopropyl) carbodiimide hydrochloride and stirred at ambient temperature for 4 h. The resulting solution was washed sequentially with 10% citric acid, aqueous NaHCO₃, and water; dried over Na₂SO₄, and concentrated in vacuo to provide 4.00 g (85%) of the desired compound.

B. (2S,3R,4R,5S)-2,5-Di-(N-(Cbz-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

A solution of 28.7 mg (0.096 mmol) of the resultant compound of Example 26 in 1 ml of dioxane was treated with 139 mg (0.40 mmol) of the resultant compound of Example 27A and stirred at ambient temperature for 24 h. The resulting solution was treated with 0.5 ml of 3N NaOH, stirred for 15 min, extracted with two portions of 10% methanol in chloroform, dried over Na₂SO₄, and concentrated in vacuo. The residue was chromatographed on silica gel using 3% methanol in chloroform to provide 42.4 mg (58%) of the desired compound (R_f 0.35, 5% methanol in chloroform) as a white solid, m.p. 231-232°C. Mass spectrum: (M + H)⁺ = 767.
Anal. Calcd for C₄₄H₅₄N₄O₈·0.25H₂O: C, 68.51; H, 7.12; N, 7.26. Found: C, 68.48; H, 7.11; N, 7.12.

Example 28

(2S,3R,4R,5S)-2,5-Di-(N-(valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 13C with the resultant compound of Example 27B provided the desired compound (R_f 0.07, 10% methanol in chloroform) as a white solid, m.p. 205-207°C. Mass spectrum: (M + H)⁺ = 499.
Anal. Calcd for C₂₈H₄₂N₄O₄·0.75H₂O: C, 65.66; H, 8.56; N, 10.94. Found: C, 65.47; H, 7.93; N, 10.59.

Example 29 (reference example)

A. N-((2-Pyridinyl)methoxycarbonyl)-valine.

Using the procedure of Example 22B but adding twice the amount of 1M HCl provided the desired compound.

Example 30 (reference example)

2,4-Di-(N-(((3-pyridinyl)carbonyl)-valinyl)-amino)-1,5-diphenyl-3-hydroxypentane.

A solution of 15 mg (0.032 mmol) of the resultant compound of Example 17 and 0.01 ml (0.09 mmol) of 4-methylmorpholine in 2 ml of dichloromethane was cooled to 0°C and treated with 12 mg (0.067 mmol) of nicotinyl chloride hydrochloride. The resulting solution was stirred at 0°C for 1 h, washed with aqueous NaHCO₃, dried over Na₂SO₄, and concentrated in vacuo. The residue was recrystallized from chloroform/ethyl acetate/hexane to afford the desired compound (R_f 0.40, 10% methanol in chloroform) as a white solid, m.p. 228-230. Mass spectrum: (M + H)⁺ = 679.

Example 31

2,5-Di-(N-(((3-pyridinyl)carbonyl)-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 30 with the resultant compound of Example 28 provided the desired compound.

Example 32

2,5-Di-(N-((3-pyridinyl)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 30 but replacing the resultant compound of Example 17 with the resultant compound of Example 26 provided the desired compound.

Example 33

2,5-Di-(N-((4-pyridinyl)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 30 but replacing the resultant compound of Example 17 with the resultant compound of Example 26 and replacing nicotinyl chloride hydrochloride with isonicotinyl chloride hydrochloride provided the desired compound.

Example 34

A. Benzyl 2-(1-morpholinyl)acetate.

A solution of 1.5 ml (17 mmol) of morpholine in 40 ml of dichloromethane was treated with 1 ml (6.3 mmol) of benzyl 2-bromoacetate. The resulting solution was stirred at ambient temperature for 16 h. The resulting solution was filtered and concentrated in vacuo. The residue was purified by silica gel chromatography using 3:1 chloroform:ethyl acetate to provide 1.35 g (91%) of the desired compound. ¹H NMR (CDCl₃) δ 2.59 (m, 4 H), 3.27 (s, 2 H), 3.77 (m, 4 H), 5.17 (s, 2 H), 7.3-7.4 (m, 5 H).

B . 2-(1-Morpholinyl)acetic Acid.

Using the procedure of Example 23B with the resultant compound of Example 34A provided the desired compound.

C. 2,5-Di-(N-((2-(1-morpholinyl)acetyl)-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

The resultant compound of Example 34B was coupled to the resultant compound of Example 28 using the coupling procedure of Example 8 provided, after silica gel chromatography using a gradient of 3-5% methanol in chloroform, the desired compound (R_f 0.31, 10% methanol in chloroform).

Example 35

A. Benzyl 2-(1-Imidazolyl)acetate.

A solution of 1.4 g (21 mmol) of imidazole and 1.0 ml (6.3 mmol) of benzyl 2-bromoacetate in 40 ml of dichloromethane was stirred at ambient temperature for 16 h. The resulting solution was washed with water, dried over Na₂SO₄, and concentrated in vacuo. Silica gel chromotography of the residue using 5% methanol in chloroform provided 1.22 g (89%) of the desired compound as an oil. ¹H NMR (CDCl₃) δ 4.73 (s, 2 H), 5.21 (s, 2 H), 6.96 (t, J = 1 Hz, 1 H), 7.11 (t, J = 1 Hz, 1 H), 7.36 (m, 5 H), 7.51 (br s, 1 H).

B. 2-(1-Imidazolyl)acetic Acid

The resultant compound of Example 35A was hydrogenolyzed according to the procedure of Example 23B except that water was added prior to filtration to solubilize the product. Removal of the solvent after filtration provided the desired compound.

C. 2,5-Di-(N-((2-(1-imidazolyl)acetyl)-valinyl-amino)-1,5-diphenyl-3,4-dihydroxypentane.

The resultant compound of Example 35B was coupled to the resultant compound of Example 28 using the carbodiimide coupling procedure described in Example 8 to give a crude mixture in which the product was soluble. The mixture was diluted with ethyl acetate, filtered, and the solid was washed sequentially with water and ethyl acetate. The residue was air-dried to provide the desired compound in 40% yield.

Example 36

A. 1-(2-Bromohexanoyl)-4-methylpiperazine.

Using the mixed anhydride procedure of Example 1F, 2-bromohexanoic acid was coupled to 1-methylpiperazine to provide the desired compound.

B. 2,5-Di-(N-((2-(4-methylpiperazin-1-yl)carbonyl)pent-1-yl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 19A but replacing the resultant compound of Example 15A with the resultant compound of Example 36A and replacing O-benzylhydroxylamine hydrochloride with the resultant compound of Example 26 provided the desired compound.

Example 37

2,5-Di-(N-(2-methoxycarbonyl-3-phenylprop-1-yl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 19A but replacing O-benzylhydroxylamine hydrochloride with the resultant compound of Example 26 provided the desired compound.

Example 38

A. 4-(2-Benzyloxycarbonyl)-3-methylprop-1-yl)-1,1-dioxo-1,4-thiazine.

According to the method of Kawaguchi, et. al. (Agric. Biol. Chem. 1987, 51, 435), 3-sulfolene was ozonolyzed and aminated with L-valine benzyl ester p-toluenesulfonate to provide the desired compound.

B. 4-(2-Carboxy-3-methylprop-1-yl)-1,1-dioxo-1,4-thiazine.

The resultant compound of Example 38A was hydrogenolyzed according to the procedure of Example 23B to provide the desired compound.

C. 2,5-Di-(N-(2-(1,1-dioxothiazin-4-yl)-3-methylbutanoyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

The resultant compound of Example 38B was coupled to the resultant compound of Example 26 using the coupling procedure described in Example 20D to provide the desired compound.

Example 39

A. 4-(2-(Benzyloxycarbonyl)-3-methylprop-1-yl)morpholine.

According to the method of Kawaguchi, et al. (Agric. Biol. Chem. 1987, 51, 435), 2,5-dihydrofuran was ozonolyzed and aminated with L-valine benzyl ester p-toluenesulfonate to provide the desired compound.

B. 4-(2-Carboxy-3-methylprop-1-yl)morpholine.

The resultant compound of Example 39A was hydrogenolyzed according to the procedure of Example 23B to provide the desired compound.

C. 2,5-Di-(N-(2-(morpholin-4-yl)-3-methylbutanoyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

The resultant compound of Example 39B was coupled to the resultant compound of Example 26 using the coupling procedure described in Example 20D to provide the desired compound.

Example 40

2,5-Di-(N-(Cbz-isoleucinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

A mixture of 10 mg (0.033. mmol) of the resultant compound of Example 26 and 38.6 mg (0.10 mmol) of Cbz-isoleucine p-nitrophenyl ester in 0.2 ml of tetrahydrofuran was stirred at ambient temperature for 21 h.. The resulting misture was diluted with 1 ml of tetrahydrofuran, treated with 0.5 ml of 3N NaOH, stirred for 45 min, extracted with chloroform, washed sequentially with 3N NaOH and saturated brine, dried over MgSO₄, and concentrated. The residue was purified on silica gel by eluting with 2% methanol in chloroform to provide 23 mg (86%) of the desired compound. Mass spectrum: (M + H)⁺ = 795.

Example 41

2,5-Di-(N-(Cbz-alaninyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 40 but replacing Cbz-isoleucine p-nitrophenyl ester with Cbz-alaninyloxy-succinimide provided the desired compound. Mass spectrum: (M + H)⁺ = 711.

Example 42

2,5-Di-(N-(Cbz-phenylalaninyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 40 but replacing Cbz-isoleucine p-nitrophenyl ester with Cbz-phenylalanine p-nitrophenyl ester provided the desired compound. Mass spectrum: (M + H)⁺ = 863.

Example 43

2,5-Di-(N-(Cbz-leucinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 40 but replacing Cbz-isoleucine p-nitrophenyl ester with Cbz-leucine p-nitrophenyl ester provided the desired compound. Mass spectrum: (M + H)⁺ = 795.

Example 44 (reference example)

A. N-((Benzyloxycarbonyl)methyl)-valine Methyl Ester.

A solution of 2.12 g (12.6 mmol) of L-valine methyl ester hydrochloride, 2.0 ml (12.6 mmol) of benzyl bromoacetate, and 3.5 ml (31 mmol) of 4-methylmorpholine in 100 ml of dioxane was heated at reflux for 4 h. After being allowed to cool, the solution was concentrated in vacuo and partitioned between ether and water. The organic layer was dried over MgSO₄ and concentrated in vacuo. Chromatography on silica gel using 20% ethyl acetate in hexane provided 0.77 g (22%) of the desired compound as a colorless oil (R_f 0.24, 20% ethyl acetate in hexane). ¹H NMR δ 0.95 (d, J = 7 Hz, 6 H), 1.96 (br, 1 H), 1.98 (octet, J = 7 Hz, 1 H), 3.08 (d, J = 6 Hz, 1 H), 3.43 (AA', 2 H), 3.71 (s, 3 H), 5.16 (s, 2 H), 7.36 (m, 5 H).

Example 45

(2S,3S,4S,5S)- and (2S,3R,4S,5S)-2,5-Diamino-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 26 with the more mobile mixture of compounds of Example 25B provided a mixture of diamines which were separated by silica gel chromatography using 2% isopropylamine in chloroform containing sequential amounts of 1%, 2% and 5% methanol.
(2S,3S,4S,5S)-2,5-Diamino-3,4-dihydroxy-1,6-diphenylhexane: R_f 0.40 (5% methanol/2% isopropylamine in chloroform), ¹H NMR (CDCl₃) δ 2.63 (dd, J = 14, 11 Hz, 2 H), 2.85 (dd, J = 14, 4 Hz, 2 H), 3.60 (dt, J = 11, 4 Hz, 2 H), 3.92 (d, J = 3 Hz, 2 H), 7.2-7.4 (m, 10 H).
(2S,3R,4S,5S)-2,5-Diamino-3,4-dihydroxy-1,6-diphenylhexane: R_f 0.23 (5% methanol/2% isopropylamine in chloroform), m.p. 115-119°C, ¹H NMR (CDCl₃) δ 2.46 (dd, J = 14, 9 Hz, 1 H), 2.61 (dd, J = 14, 11 Hz, 1 H), 3.02 (td, J = 9, 3 Hz, 1 H), 3.19 (dd, J = 14, 4 Hz, 1 H), 3.35-3.4 (m, 2 H), 3.51 (t, J = 9 Hz, 1 H), 3.76 (dd, J = 9, 3 Hz, 1 H), 7.2-7.4 (m, 10 H).

Example 46

(2S,3S,4S,5S)-2,5-Di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

A solution of 15 mg (0.05 mmol) of (2S,3S,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane in 0.5 ml of dichloromethane was treated with 24 mg (0.11 mmol) of di-t-butyldicarbonate and stirred at ambient temperature. After 16 h, the solution was concentrated in vacuo, and the residue was purified by silica gel chromatography using 30% ethyl acetate in hexane to provide 17 mg (68%) of the desired compound, m.p. 216-218°C. ¹H NMR δ 1.40 (s, 18 H), 2.97 (dd, J = 14, 5 Hz, 2 H), 3.20 (dd, J = 14, 5 Hz, 2 H), 3.22 (m, 2 H), 4.03 (m, 2 H), 4.35 (d, J = 5 Hz, 2 H), 4.41 (d, J = 9 Hz, 2 H), 7.2-7.3 (m, 10 H). Mass spectrum: (M + H)⁺ = 501.

Example 47

(2S,3R,4S,5S)-2,5-Di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 46 with (2S,3S,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane provided the desired compound (R_f 0.32, 30% ethyl acetate in hexane), as a white solid, m.p. 208-212°C. ¹H NMR δ 1.33 (s, 9 H), 1.40 (s, 9 H), 2.67 (m, 1 H), 2.75-2.95 (m, 6 H), 3.47 (m, 2 H), 4.14 (m, 2 H), 4.58 (m, 1 H), 4.83 (br d, 1 H), 4.93 (br d, 1 H), 7.15-7.3 (m, 10 H).

Example 48

(2S,3R,4S,5S)-2,5-Di-(N-(Cbz-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 27B with 25 mg (0.083 mmol) of (2S,3R,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane provided the desired compound (31%, R_f 0.3, 5% methanol in chloroform) as a white solid, m.p. 230-234°C. Mass spectrum: (M + H)⁺ = 767.

Example 49

(2S,3S,4S,5S)-2,5-Di-(N-(Cbz-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Cbz-valine p-nitrophenyl ester was coupled to (2S,3S,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane using the procedure of Example 27B to provide the desired compound (R_f 0.42, 5% methanol in chloroform) as a white solid, m.p. 239-242°C in 86 % yield.

Example 50

(2S,3R,4R,5S)-2,5-Di-(N-((t-butyloxy)carbonyl)amino)-1,6-dicyclohexyl-3,4-dihydroxyhexane.

A mixture of 180 mg (0.36 mmol) of (2S,3R,4R,5S)-2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane and 180 mg of 5% rhodium on carbon in 50 ml of methanol was shaken under 4 atmospheres of hydrogen for 24 h. The resulting mixture was filtered and'concentrated in vacuo. The residue was purified by silica gel chromatography using 5% ethyl acetate in hexane to provide 120 mg (65%) of the desired compound (R_f 0.35, 30% ethyl acetate in hexane) as a white solid, m.p. 224-226°C. Mass spectrum: (M + H)⁺ = 513.
Anal. Calcd for C₂₈H₅₂N₂O₆: C, 65.69; H, 10.22; N, 5.46. Found: C, 65.27; H, 10.16; N, 5.40.

Example 51

(2S,3S,4R,5S)-5-Amino-2-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

A solution of 200 mg (0.67 mmol) of (2S,3S,4R,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane in 10 ml of dichloromethane was treated with 174 mg (0.8 mmol) of di-t-butyldicarbonate. After being allowed to stir overnight at ambient temperature, the solution was concentrated, and the residue was purified by silica gel chromatography using 10% methanol in chloroform to provide 180 mg (56%) of the desired compound along with 80 mg (20%) of the resultant compound of Example 47

Example 52

(2S,3S,4R,5S)-2-(N-((t-Butyloxy)carbonyl)amino)-5-(N-(Cbz-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

The resultant compound of Example 51 was coupled to Cbz-valine using the carbodiimide coupling procedure of Example 8 to provide the desired compound (R_f 0.48, 5% methanol in chloroform) as a white solid, m.p. 178-182°C, in 88% yield. Mass spectrum: (M + H)⁺ = 634.
Anal. Calcd for C₃₆H₄₇N₃O₇·0.5H₂O: C, 67.27; H, 7.53; N, 6.54. Found: C, 67.18; H, 7.45; N, 6.71.

Example 53

A. N,N-Dimethylvaline.

A mixture of 2.5 g of L-valine, 0.5 g of 10% palladium on carbon, in 93 ml of methanol and 7 ml of formalin was shaken under 4 atmospheres of hydrogen. After 24 h, the solution was filtered and concentrated in vacuo to provide the crude desired compound.

B. (2S,3R,4R,5S)-2,5-Bis-(N-(N,N-dimethylvalinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

The resultant compound of Example 53A was coupled to the resultant compound of Example 26 using the carbodiimide coupling procedure of Example 8 to provide the desired compound (20%, R_f 0.3, 10% methanol in chloroform) as a white solid, m.p. 200-204°C. Mass spectrum: (M + H)⁺ = 555.

Example 54

A. N-((2-Pyridinyl)methoxycarbonyl)-valine p-Nitrophenyl Ester.

A solution of 0.87 mmol of the resultant compound of Example 29A and 133. mg (0.96 mmol) of p-nitrophenol in 4 ml of tetrahydrofuran and 2 ml of dimethylformamide was treated with 183 mg (0.96 mmol) of N-ethyl-N'-(dimethylaminopropyl) carbodiimide hydrochloride and stirred at ambient temperature. After 4 h, the solvent was removed in vacuo, and the residue was partially purified by silica gel chromatography using 20% ethyl acetate in chloroform to give 0.34 mg of the desired compound contaminated with excess p-nitrophenol.

B. (2S,3R,4R,5S)-2,5-Di-(N-((2-pyridinyl)methoxycarbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

A solution of 230 mg of the crude resultant compound of Example 54A and 70 mg (0.23 mmol) of the resultant compound of Example 26 in 1 ml of 1:1 tetrahydrofuran/ dimethylformamide was stirred at ambient temperature for 16 h. The resulting solution was treated with aqueous NaHCO₃, stirred for 1 h, diluted with 5% methanol in chloroform, washed with aqueous NaHCO₃ until the washes were colorless, dried over Na₂SO₄, and concentrated. Chromatography on silica gel using 2% methanol in chloroform followed by 5% methanol in chloroform provided 140.6 mg (80%) of the desired compound (R_f 0.32, 10% methanol in chloroform) as a white solid, m.p. 196-200°C. Mass spectrum: (M + H)⁺ = 769.
Anal. Calcd for C₄₂H₅₂N₆O₈: C, 65.61; H, 6.82; N, 10.93. Found: C, 65.68; H, 6.93; N, 10.95.

Example 55

(2S,3S,4S,5S)-2,5-Di-(N-((2-pyridinyl)methoxycarbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 54B but replacing the resultant compound of Example 26 with (2S,3S,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane provided, after silica gel chromatography using a gradient of 2-5% methanol in chloroform, the desired compound (R_f 0.32, 5% methanol in chloroform) as a white solid, m.p. 220-223°C, in 79% yield. Mass spectrum: (M + H)⁺ = 769.
Anal. Calcd for C₄₂H₅₂N₆O₈·0.5H₂O: C, 64.85; H, 6.87; N, 10.80. Found: C, 64.69; H, 6.84; N, 10.63.

Example 56

(2S,3R,4S,5S)-2,5-Di-(N-((2-pyridinyl)methoxycarbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 54B but replacing the resultant compound of Example 26 with (2S,3R,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane provided, after silica gel chromatography using a gradient of 2-5% methanol in chloroform, the desired compound (R_f 0.23, 5% methanol in chloroform) as a white solid, m.p. 238-240°C, in 82% yield. Mass spectrum: (M + H)⁺ = 769.
Anal. Calcd for C₄₂H₅₂N₆O₈·0.25H₂O: C, 65.23; H, 6.84; N, 10.87. Found: C, 65.01; H, 6.89; N, 10.92.

Example 57

A. 2-(N-(t-Butyloxycarbonyl)aminomethyl)pyridine.

A solution of 21.2 g (97 mmol) of di-t-butyldicarbonate in 200 ml of dichloromethane was cooled to 0°C and treated in portions with 10 ml (97 mmol) of 2-(aminomethyl)pyridine. After being allowed to warm to ambient temperature and stirred overnight, the resulting solution was diluted with 100 ml of dichloromethane, washed with three 100 ml portions of water, dried over Na₂SO₄, and concentrated in vacuo to provide 19.8 g (98%) of the desired compound (R_f 0.28, 5% methanol in chloroform). ¹H NMR (CCDl₃) δ 1.47 (s, 9 H), 4.45 (d, J = 6 Hz, 2 H), 5.56 (br, 1 H), 7.18 (m, 1 H), 7.28 (d, J = 8 Hz, 1 H), 7.66 (td, J = 7, 2 Hz, 1 H), 8.53 (m, 1 H).

B. 2-((N-(t-Butyloxycarbonyl)-N-methylamino)methyl)pyridine.

A solution of 19.8 g (95 mmol) of the resultant compound of Example 57A in anhydrous tetrahydrofuran was cooled under N₂ atmosphere to 0°C and treated with 4.95 g (124 mmol) of sodium hydride (60% dispersion in oil). The solution was stirred for 15 min, treated dropwise with 7.1 ml (114 mmol) of methyl iodide, stirred at ambient temperature for 2 h, and quenched cautiously with water. The resulting mixture was partitioned between ether and water, dried over Na₂SO₄, and concentrated.
Chromatography on silica gel provided 14.9 g (70%) of the desired compound as a colorless oil.

C. 2-(N-methylamino)methyl)pyridine Dihydrochloride.

The resultant compound of Example 57B (3.05 g, 13.7 mmol) was treated with 30 ml of 4N HCl in dioxane and heated at 40°C for 0.5 h. The solvent was removed in vacuo to provide the crude desired compound as a light brown solid.

D. N-((N-Methyl-N-((2-pyridinyl)methyl)amino)carbonyl)-valine Methyl Ester.

A mixture of 1.61 g (7.2 mmol) of the resultant compound of Example 57C and 1.14 g (7.2 mmol) of the resultant compound of Example 20A in 40 ml of dichloromethane was treated with 2 ml (18 mmol) of 4-methylmorpholine. After being stirred for 2 h, the solution was partitioned between dichloromethane and water, dried over Na₂SO₄, and concentrated.
Chromatography on silica gel using 2% methanol in chloroform provided 1.94 g (96%) of the desired compound (R_f 0.32, 5% methanol in chloroform) as a colorless oil. ¹H NMR (CCDl₃) δ 0.93 (d, J = 7 Hz, 3 H), 0.97 (d, J = 7 Hz, 3 H), 2.16 (m, 1 H), 3.03 (s, 3 H), 3.72 (s, 3 H), 4.43 (dd, J = 8, 5 Hz, 1 H), 4.55 (s, 2 H), 6.15 (br, 1 H), 7.22 (dd, J = 8, 6 Hz, 1 H), 7.28 (d, J = 6 Hz, 1 H), 7.69 (br t, 1 H), 8.55 (d, J = 5 Hz, 1 H).

E. N-((N-Methyl-N-((2-pyridinyl)methyl)amino)carbonyl)-valine p-Nitrophenyl Ester.

Using the procedures of Example 29A and Example 54A but replacing the resultant compound of Example 22A with the resultant compound of Example 57D provided the desired compound.

F. (2S,3R,4R,5S)-2,5-Di-(N-((N-Methyl-N-((2-pyridinyl)methyl)amino)carbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 54B but replacing the resultant compound of Example 54A with the resultant compound of Example 57E provided, after silica gel chromatography using a gradient of 2-5% methanol in chloroform, the desired compound (R_f 0.28, 5% methanol in chloroform) as a white solid, m.p. 108-111°C, in 85% yield. Mass spectrum: (M + H)⁺ = 795.
Anal. Calcd for C₄₄H₅₈N₈O₆·1.25H₂O: C, 64.65; H, 7.46; N, 13.71. Found: C, 64.35; H, 7.06; N, 13.58.

Example 58

(2S,3S,4S,5S)-2,5-Di-(N-((N-Methyl-N-((2-pyridinyl)methyl)amino)carbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 54B but replacing the resultant compound of Example 54A with the resultant compound of Example 57E and replacing the resultant compound of Example 26 with (2S,3S,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane provided, after silica gel chromatography using a gradient of 1-5% methanol in chloroform, the desired compound (R_f 0.38, 5% methanol in chloroform) as a white solid, m.p. 110-112°C, in 75% yield. Mass spectrum: (M + H)⁺ = 795.
Anal. Calcd for C₄₄H₅₈N₈O₆·H₂O: C, 65.00; H, 7.44; N, 13.78. Found: C, 64.61; H, 7.21; N, 13.60.

Example 59

(2S,3R,4S,5S)-2,5-Di-(N-((N-Methyl-N-((2-pyridinyl)methyl)amino)carbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 54B but replacing the resultant compound of Example 54A with the resultant compound of Example 57E and replacing the resultant compound of Example 26 with (2S,3R,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane provided, after silica gel chromatography using a gradient of 1-2% methanol in chloroform, the desired compound (R_f 0.36, 5% methanol in chloroform) as a white solid, m.p. 159-162°C, in 79% yield. Mass spectrum: (M + H)⁺ =795.
Anal. Calcd for C₄₄H₅₈N₆O₈: C, 66.48; H, 7.35; N, 14.09. Found: C, 66.31; H, 7.43; N, 13.95.

Example 60

(2S,3R,4R,5S)-2,5-Di-(N-(((2-pyridinyl)carbonyl)-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Picolinic acid was coupled to the resultant compound of Example 28 using the carbodiimide coupling procedure described in Example 8 to provide after silica gel chromatography using a gradient of 5-10% methanol in chloroform, the desired compound (R_f 0.16, 10% methanol in chloroform) as a white solid, m.p. 167-171°C, in 61% yield. Mass spectrum: (M + H)⁺ = 709.
Anal. Calcd for C₄₀H₄₈N₆O₆: C, 67.78; H, 6.83; N, 11.86. Found: C, 67.81; H, 6.59; N, 11.78.

Example 61

A. 3-(3-Pyridinyl)propanoic Acid

A mixture of 3 g (20 mmol) of 3-(3-pyridinyl)acrylic acid and 0.3 g of 10% palladium on carbon in 150 ml of ethyl acetate was shaken under 4 atmospheres of hydrogen for 24 h. After filtration, the resulting solution was concentrated in vacuo to provide the desired compound.

B. (2S,3R,4R,5S)-2,5-Di-(N-(3-(3-pyridinyl)propanoyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

The resultant compound of Example 61A was coupled to the resultant compound of Example 28 using the carbodiimide coupling procedure described in Example 8 to provide after silica gel chromatography using a gradient of 5-10% methanol in chloroform, the desired compound (R_f 0.1, 10% methanol in chloroform) as a white solid, m.p. 260-263°C, in 37% yield. Mass spectrum: (M + H)⁺ = 765.

Example 62

(2S,3R,4R,5S)-3,4-Dihydroxy-2,5-di-(N-(N-(2-pyridyl-acetyl))-valinyl)-amino-1,6-diphenylhexane.

(2-Pyridyl)acetic acid was coupled to the resultant compound of Example 28 using the carbodiimide coupling procedure described in Example 8 to provide after silica gel chromatography using a gradient of 2-5% methanol in chloroform, the desired compound (41%, R_f 0.21, 5% methanol in chloroform) as a white solid, m.p. 208-213°C. Mass spectrum: (M + H)⁺ = 737.

Example 63

A. N-((4-Pyridinyl)methoxycarbonyl)-valine p-Nitrophenyl Ester.

Using the procedures of Example 29A and Example 54A but replacing the resultant compound of Example 22A with the resultant compound of Example 20B provided the desired compound.

B. (2S,3R,4R,5S)-2,5-Di-(N-((4-pyridinyl)methoxycarbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 54B but replacing the resultant compound of Example 54A with the resultant compound of Example 63A provided, after silica gel chromatography using a gradient of 2-5% methanol in chloroform, the desired compound (R_f 0.11, 10% methanol in chloroform) as a white solid, m.p. 221-224°C in 48% yield. Mass spectrum: (M + H)⁺ = 769.
Anal. Calcd for C₄₂H₅₂N₆O₈·0.5H₂O: C, 64.85; H, 6.87; N, 10.80. Found: C, 64.91; H, 6.81; N, 10.80.

Example 64

(2S,3R,4S,5S)-2,5-Di-(N-(t-butylaminocarbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

A solution of 30 mg (0.1 mmol) of (2S,3R,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane in 1 ml of dichloromethane was treated with 25 µl (0.22 mmol) of t-butylisocyanate. The resulting solution was stirred at ambient temperature, diluted with dichloromethane, washed with brine, dried over Na₂SO₄, and concentrated in vacuo. Silica gel chromatography using a gradient of 1-3% methanol in chloroform provided 49 mg (98%) of the desired compound (R_f 0.4, 10% methanol in chloroform) as a white solid, m.p. 193-196°C. Mass spectrum: (M + H)⁺ = 499.
Anal. Calcd for C₂₈H₄₂N₄O₄·H₂O: C, 65.09; H, 8.58; N, 10.84. Found: C, 65.17; H, 8.21; N, 10.77.

Example 65

(2S,3R,4S,5S)-2,5-Di-(N-(isopropylaminocarbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 64 but replacing t-butylisocyanate with isopropylisocyanate provided the desired compound (R_f 0.24, 10% methanol in chloroform) as a white solid, m.p. 220-222°C, in 81% yield. Mass spectrum: (M + H)⁺ = 471.
Anal. Calcd for C₂₆H₃₈N₄O₄·0.25H₂O: C, 65.73; H, 8.17; N, 11.79.

Example 66

(2S,3S,4S,5S)-2,5-Di-(N-((4-pyridinyl)methoxycarbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 54B but replacing the resultant compound of Example 54A with the resultant compound of Example 63A and replacing the resultant compound of Example 26 with (2S,3S,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane provided, after silica gel chromatography using a gradient of 2-5% methanol in chloroform, the desired compound (35%, R_f 0.25, 10% methanol in chloroform) as a white solid, m.p. 190-193°C. Mass spectrum: (M + H)⁺ = 769.

Example 67

A. N-((3-Pyridinyl)methoxycarbonyl)-valine p-Nitrophenyl Ester.

Using the procedures of Example 29A and Example 54A but replacing the resultant compound of Example 22A with the resultant compound of Example 21A provided the desired compound.

B. (2S,3S,4S,5S)-2,5-Di-(N-((3-pyridinyl)methoxycarbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 54B but replacing the resultant compound of Example 54A with the resultant compound of Example 67A and replacing the resultant compound of Example 26 with (2S,3S,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane provided, after silica gel chromatography using a gradient of 5-10% methanol in chloroform, the desired compound (R_f 0.31, 10% methanol in chloroform) as a white solid, m.p. 202-207°C. Mass spectrum: (M + H)⁺ = 769.

Example 68

(2S,3R,4S,5S)-2,5-Di-(N-((3-pyridinyl)methoxycarbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 54B but replacing the resultant compound of Example 54A with the resultant compound of Example 67A and replacing the resultant compound of Example 26 with (2S,3R,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane provided, after silica gel chromatography using a gradient of 2-5% methanol in chloroform, the desired compound (31%, R_f 0.28, 10% methanol in chloroform) as a white solid, m.p. 212-216°C. Mass spectrum: (M + H)⁺ = 769.

Example 69

(2S,3S,4R,5S)-2-(N-((t-Butyloxy)carbonyl)amino)-5-(N-((2-pyridinyl)methoxycarbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 54B but replacing the resultant compound of Example 26 with the resultant compound of Example 51 provided, after silica gel chromatography using a gradient of 0-2% methanol in chloroform, the desired compound (R_f 0.57, 5% methanol in chloroform) as a white solid, m.p. 202-204°C, in 61% yield. Mass spectrum: (M + H)⁺ = 635.

Example 70

(2S,3S,4R,5S)-2-Amino-5-(N-((3-pyridinyl)methoxycarbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

The resultant compound of Example 69 (200 mg, 0.31 mmol) was treated with 20 ml of 4N HCl in dioxane. After being stirred at ambient temperature for 2 h, the solvent was removed in vacuo. The residue was partitioned between chloroform and aqueous NaHCO₃, dried over Na₂SO₄, and concentrated. Silica gel chromatography using a gradient of 2% methanol/2% isopropylamine in chloroform provided 140 mg (84%) of the desired compound.

Example 71

(2S,3S,4R,5S)-2-(N-Succinylamino)-5-(N-((3-pyridinyl)methoxycarbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

A solution of 50 mg (0.93 mmol) of the resultant compound of Example 70 in 0.5 ml of dichloromethane was treated with 9.3 mg (0.93 mmol) of succinic anhydride. The resulting mixture was stirred overnight at ambient temperature and concentrated in vacuo to a solid which was washed with chloroform. The solvent was decanted to provide the desired compound (R_f 0.91, 1:1:1:1 ethyl acetate/n-butanol/water/acetic acid) as a white solid. Mass spectrum: (M + H)⁺ = 635.

Example 72

A. N-(Chlorosulfonyl)-valine Methyl Ester.

A suspension of 15.3 g (90 mmol) of L-valine methyl ester hydrochloride in 45 ml of acetonitrile was treated with 22 ml (270 mmol) of sulfuryl chloride and heated at reflux for 16 h. The resulting light yellow solution was allowed to cool and concentrated in vacuo to a viscous oil. The oil was treated two times with acetonitrile followed each time by concentration in vacuo. The crude desired product was thus obtained as a viscous oil.

B. N-((N-Methyl-N-((2-pyridinyl)methyl)amino)sulfonyl)-valine Methyl Ester.

A mixture of 13.7 mmol of the resultant compound of Example 57C and 3.17 g (13.7 mmol) of the resultant compound of Example 72A in 100 ml of dichloromethane was cooled to 0°C and treated with 6 ml of 4-methylmorpholine. The resulting solution stirred for 2 h, diluted with dichloromethane, washed with aqueous NaHCO₃, dried over Na₂SO₄, and concentrated in vacuo. Chromatography on silica gel using 30% ethyl acetate in chloroform provided 1.72 g (40%) of the desired compound as a colorless oil. ¹H NMR (CCDl₃, major rotamer) δ 0.95 (d, J = 7 Hz, 3 H), 1.03 (d, J = 7 Hz, 3 H), 2.12 (m, 1 H), 2.79 (s, 3 H), 3.76 (s, 3 H), 3.95 (dd, J = 8, 4 Hz, 1 H), 4.54 (AA', 2 H), 6.40 (d, J = 8 Hz, 1 H), 7.26 (m, 1 H), 7.35 (d, 6 Hz, 1 H), 7.71 (br t, 1 H), 8.59 (d, J = 4 Hz, 1 H). Mass spectrum: (M + H)⁺ = 316.

C. N-((N-Methyl-N-((2-pyridinyl)methyl)amino)sulfonyl)-valine.

A solution of 200 mg (0.63 mmol) of the resultant compound of Example 72B in 2.5 ml of dioxane was treated with 2.5 ml of 0.5M LiOH. After being stirred overnight at ambient temperature, the solution was concentrated in vacuo at 30°C, diluted with dioxane and water, neutralized with 1M HCl, and concentrated in vacuo to provide the crude desired compound.

D. (2S,3R,4R,5S)-2,5-Di-(N-((N-Methyl-N-((2-pyridinyl)methyl)amino)sulfonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

The resultant compound of Example 72C was coupled to the resultant compound of Example 26 using the carbodiimide coupling procedure of Example 8 to provide, after silica gel chromatography using 2% methanol in chloroform, the desired compound (45%, R_f 0.5, 10% methanol in chloroform) as a white solid, m.p. 85-89°C. Mass spectrum: (M + H)⁺ = 867.
Anal. Calcd for C₄₂H₅₈N₈O₈S₂·H₂O: C, 57.00; H, 6.83; N, 12.66. Found: C, 56.78; H, 6.56; N, 12.45.

Example 73

A. (4S)4-Benzyl-3-(3-methylbutanoyl)oxazolidine-2-one.

Using the procedure of Example 14A but replacing 4-(2-propyl)-oxazolidine-2-one with 4-benzyloxazolidine-2-one and replacing 4-methylpentanoyl chloride with isovaleryl chloride provided the desired compound.

B. (4S,2'S)-3-(2-(t-Butyloxycarbonyl)methyl-4-methylbutanoyl)-4-benzyloxazolidine-2-one.

Using the procedure of Example 14B with the resultant compound of Example 73A provided, after silica gel chromatography using 15% ethyl acetate in hexane followed by dichloromethane, the desired compound (R_f 0.35, 20% ethyl acetate in hexane) in 88% yield.

C. Benzyl (2S)-2-(t-Butyloxycarbonyl)methyl-4-methylbutanoate.

Using the procedure of Example 14C with the resultant compound of Example 73B provided, after silica gel chromatography using 6% ethyl acetate in hexane, the desired compound (R_f 0.43, 10% ethyl acetate in hexane) in 60% yield.

D. Benzyl (2S)-2-Carboxymethyl-4-methylbutanoate.

Using the procedure of Example 14D with the resultant compound of Example 73C provided the desired compound as a crude colorless oil.

E. Benzyl (2S)-2-(((N-Methyl-N-((2-pyridinyl)methyl)amino)carbonyl)methyl)-4-methylbutanoate.

The resultant compound of Example 73D was coupled to the resultant compound of Example 57C using the mixed anhydride coupling method described in Example 1F to provide, after silica gel chromatography using 60% ethyl acetate in chloroform, the desired compound (R_f 0.19, 60% ethyl acetate in chloroform) in 73% yield.

F. (2S)-2-(((N-Methyl-N-((2-pyridinyl)methyl)amino)-carbonyl)methyl)-4-methylbutanoic Acid.

The resultant compound of Example 73E was hydrogenolyzed according to the procedure described in Example 13C to provide the desired compound.

G. (2S,3R,4R,5S,2'S,2"S)-2,5-Di-(2-(((N-methyl-N-((2-pyridinyl)methyl)amino)carbonyl)methyl)-4-methylbutanoyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

The resultant compound of Example 73F was coupled to the resultant compound of Example 26 using the carbodiimide coupling procedure of Example 8 to provide, after silica gel chromatography using 5% methanol in chloroform, the desired compound in 42% yield, m.p. 169-170°C. Mass spectrum: (M + H)⁺ = 793.
Anal. Calcd for C₄₆H₆₀N₆O₆·0.5H₂O: C, 68.89; H, 7.67; N, 10.48. Found: C, 68.85; H, 7.80; N, 10.16.

Example 74

Ethyl 3-(2-Pyridinyl)acrylate.

A suspension of 0.43 g (10.7 mmol) of sodium hydride (60% dispersion in oil) in 60 ml of anhydrous tetrahydrofuran was cooled to 0°C and treated with 2.1 ml (10.5 mmol) of triethylphosphonoacetate. The resulting solution was stirred for 10 min, treated with 1.0 ml (10.5 mmol) of pyridine-2-carboxaldehyde, heated at reflux for 2 h, and stirred overnight at ambient temperature. The resulting mixture was partitioned between ether and aqueous ammonium chloride, washed sequentially with water and saturated brine, dried over MgSO₄, and concentrated in vacuo. Chromatography on silica gel using 25% ethyl acetate in hexane provided 1.54 g (83%) of the desired compound as a colorless liquid. ¹H NMR (CDCl₃) δ 1.34 (t, J = 7 Hz, 3 H), 4.28 (q, J = 7 Hz, 2 H), 6.92 (d, J = 16 Hz, 1 H), 7.27 (ddd, J = 8, 5, 1 Hz, 1 H), 7.43 (d, J = 8 Hz, 1 H), 7.72 (m, 1 H), 8.65 (m, 1 H).

B. 3-(2-Pyridinyl)acrylic Acid.

The resultant compound of Example 74A was hydrolyzed according to the procedure of Example 29A to provide the desired compound.

C. (2S,3R,4R,5S)-2,5-Di-(N-(3-(2-pyridinyl)propenoyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedures of Example 61A and Example 61B but replacing 3-(3-pyridinyl)acrylic acid with the resultant compound of Example 74B provided the desired compound.

Example 75

(2S,3R,4R,5S)-2,5-Di-(N-(3-(2-pyridinyl)propanoyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedures of Example 61A and Example 61B but replacing 3-(3-pyridinyl)acrylic acid with 3-(2-pyridinyl)acrylic acid provided the desired compound (R_f 0.21, 10% methanol in chloroform). Mass spectrum: (M + H)⁺ = 765.

Example 76

(2S,3R,4R,5S)-2,5-Di-(N-((3-pyridinyl)methoxycarbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 20D, the resultant compound of Example 21B was coupled to the resultant compound of Example 26 to provide, after silica gel chromatography using 5% methanol in chloroform, the desired compound (R_f 0.25, 10% methanol in chloroform) as a white solid, m.p. 207-208°C, in 32% yield. Mass spectrum: (M + H)⁺ = 769.
Anal. Calcd for C₄₂H₅₂N₆O₈·1.25H₂O: C, 63.74; H, 6.94; N, 10.62. Found: C, 63.70; H, 6.70; N, 10.54.

Example 77

(2S,3R,4S,5S)-2,5-Di-(N-((2-(4-morpholinyl)ethyloxy)-carbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

The resultant compound of Example 24B was coupled to the resultant compound of Example 26 using the carbodiimide coupling procedure of Example 20D to provide, after silica gel chromatography using 5% methanol in chloroform, the desired compound (R_f 0.21, 10% methanol in chloroform) as a white solid, m.p. 227-230°C (dec) in 47% yield. Mass spectrum: (M + H)⁺ = 813.
Anal. Calcd for C₄₂H₆₄N₆O₁₀·1.25H₂O: C, 60.38; H, 8.02; N, 10.06. Found: C, 60.21; H, 7.78; N, 10.43.

Example 78

(2S,3S,4R,5S)-2-(N-((t-Butyloxy)carbonyl)amino)-5-(N-((3-pyridinyl)methoxycarbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 20D the resultant compound of Example 21B was coupled to the resultant compound of Example 51 to provide, after silica gel chromatography using 3% methanol in chloroform, the desired compound (R_f 0.21, 5% methanol in chloroform) as a white solid, m.p. 175-178°C, in 66% yield. Mass spectrum: (M + H)⁺ = 635.
Anal. Calcd for C₃₅H₄₆N₄O₂·0.75H₂O: C, 64.65; H, 7.38; N, 8.64. Found: C, 64.61; H, 7.16; N, 8.80.

Example 79

(2S,3R,4S,5S)-2,5-Di-(N-((N-Methyl-N-((2-pyridinyl)methyl)amino)sulfonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

The resultant compound of Example 72C was coupled to (2S,3R,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane using the carbodiimide coupling procedure of Example 8 to provide, after silica gel chromatography, the desired compound (R_f 0.65, 10% methanol in chloroform) as a white solid, m.p. 91-93°C. Mass spectrum: (M + H)⁺ = 867.
Anal. Calcd for C₄₂H₅₈N₈O₈S₂·H₂O: C, 57.00; H, 6.83; N, 12.66. Found: C, 67.26; H, 8.30; N, 4.98.

Example 80

(2S,3S,4S,5S)-2,5-Di-(N-((N-methyl-N-((2-pyridinyl)methyl)amino)sulfonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

The resultant compound of Example 72C was coupled to (2S,3S,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane using the carbodiimide coupling procedure of Example 8 to provide, after silica gel chromatography, the desired compound (R_f 0.54, 10% methanol in chloroform) as a white solid, m.p. 75-77°C. Mass spectrum: (M + H)⁺ = 867.

Example 81

(2S,3S,4S,5S,2'S,-2"S)-2,5-Di-((2-hydroxy-3-methylpentanoyl)-amino)-3,4-dihydroxy-1,6-diphenylhexane.

L-2-Hydroxy-3-methylvaleric acid was coupled to the resultant compound of Example 26 using the diimide coupling procedure described in Example 8 to provide the desired compound (R_f 0.23, 10% methanol in chloroform) as a white solid, m.p.226-230°C.
Anal. Calcd for C₃₀H₄₄N₂O₆·0.5H₂O: C, 67.01; H, 8.44; N, 5.21. Found: C, 67.26; N, 8.30; N, 4.98.

Example 82

A. 2-(t-Butyloxycarbonyl)methyl-3-methylbutanoic Acid

The resultant compound of Example 73B was hydroyzed according to the procedure of Example 1D except that excess 30% hydrogen peroxide was included in the reaction mixture to provide the crude desired compound.

B. (2S,3S,4R,5S)-2-(N-(2-(t-Butyloxycarbonyl)methyl-3-methylbutanoyl)amino)-5-(N-((3-pyridinyl)methoxycarbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

The resultant compound of Example 82A was coupled to the resultant compound of Example 70 using the carbodiimide coupling procedure of Example 8 to provide, after silica gel chromatography using a gradient of 1-5% methanol in chloroform, the desired compound in 22% yield.

B. (2S,3S,4R,5S)-2-(N-(2-Carboxymethyl-3-methylbutanoyl)amino)-5-(N-((3-pyridinyl)methoxycarbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane Trifluoroacetate Salt.

The resultant compound of Example 82B (45 mg) was treated with 1.5 ml of 2:1 dichloromethane/trifluoroacetic acid. After 4 h at ambient temperature, the solution was concentrated in vacuo to provide 28 mg (55%) of the desired compound as an off-white solid, m.p. 208-210°C.

Example 83

(2S,3S,4S,5S,2'S,2"S)-2,5-Di-((2-((methoxy)-methoxy)-3-methylpentanoyl)-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 81 but replacing L-2-hydroxy-3-methylvaleric acid with L-2-((methoxy)-methoxy)-3-methylvaleric acid provided, after silica gel chromatography using 20% ethyl acetate in hexane, the desired compound (88%, R_f 0.18, 20% ethyl acetate in hexane) as a white solid, m.p. 190-194°C. Mass spectrum: (M + H)⁺ = 617.

Example 84 (reference example)

A. (6S,7R,8R,9S)-6,9-Di-((N-toluenesulfonyl)amino)-7,8-O-isopropylidenetetradecanediol

To a solution of 1.175 g (5.715 mmol) of cuprous bromide-dimethylsufide complex in 8 mL of anhydrous, oxygen-free THF at -40°C was added 4.57 mL (11.43 mmol) of 2.5 M n-butyl lithium. The solution was stirred for 0.5 h at -40°C and then cooled to -60°C. A solution of (2S,3R,4R,5S)-1,2:5,6-diimino-3,4-O-isopropylidenehexanediol (703 mg, 1.43 mmol(Y.L. Merrer, et al, Heterocycles, 1987, 25, 541-548)) in -16 mL of anhydrous, oxygen-free THF was added. The solution was allowed to warm slowly to approximately -25°C and stirred for 4 h at that temperature. The reaction mixture was treated with 10% concentrated ammonium hydroxide in saturated ammonium chloride solution and the aqueous mixture was extracted with diethyl ether. The organic solution was washed with brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography on a 1.0 X 27 cm silica gel column eluted with 20% ethyl acetate in hexane to give 0.414 g (47.6% yield) of the title compound; DCI/NH₃ MS M/Z: 626 (M+NH₄)⁺; The 300 MHz ¹H NMR spectrum was consistent with the assigned structure.

B. N,N-Bis-(Cbz-valinyl)-(2S, 3R, 4R, 5S)-6,9-diamino-7,8-O-isopropylidenetetradecanediol

The resultant compound of Example 84A in 3 mL of anhydrous diethyl ether was added to -75 mL of liquid ammonia and small pieces of sodium metal were added until a blue color persisted for 5 minutes. The reaction was quenched with ammonium chloride and the ammonia was evaporated. The residue was dissolved in diethyl ether and the ether solution was washed with dilute aqueous ammonium hydroxide and brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the intermediate 6,9-diamino compound. The diamine (159 mg, 0.53 mmol) was dissolved in 5 mL of THF and the THF solution was cooled in an ice bath. To the cooled solution was added 532 mg (2.12 mmol) of N-carbobenzyloxyvaline, followed by 406 mg (2.12 mmol) of N-ethyl-N'-(dimethylaminopropyl)carbodiimide and 295 µL (2.12 mmol) of triethylamine. The reaction mixture was allowed to gradually warm to ambient temperature and was stirred at ambient temperature overnight. The solvent which had evaporated from the reaction mixture was replaced with 5 mL of THF and the mixture was stirred for 2 h. The reaction mixture was then diluted with ethyl acetate and washed with saturated aqueous sodium bicarbonate solution and brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on a 1.5 X 35 cm silica gel column eluted with 30% ethyl acetate to to give 83 mg (20% yield) of the title compound; DCI/NH₃ MS M/Z: 784 (M+NH₄)⁺, 767 (M+H)⁺; The 300 MHz ¹H NMR spectrum is consistent with the assigned structure. Analysis calculated for C₄₃H₆₆N₄O₈: C, 67.36; H, 8.62; N, 7.31. Found: C, 67.68; H, 9.02; N, 7.27.

C. N,N-Bis-(Cbz-valinyl)-(6S, 7R, 8R, 9S)-6,9-diamino-7,8-tetradecanediol

A solution of the resultant compound of Example 84B (80 mg, 0.1 mmol) in 3.0 mL of trifluoroacetic acid containing 0.3 mL of water was stirred at 0°C for 9.75 h. The solvent was evaporated under reduced pressure. The residue was purified by flash chromatography on a 1.0 X 22 cm silica gel column eluted with 50% ethyl acetate in hexane to give 51 mg (45% yield) of the title compound; ¹H NMR (CDCl₃) δ 0.86 (t, 6H), 0.92 (d, 6H), 0.98 (d, 6H), 1.20-1.33 (br m, 12H), 1.51-1.61 (br m, 2H), 2.12-2.22 (m, 2H), 3.39 (br s, 1H), 3.55 (br s, 1H), 3.82-3.92 (m, 2H), 3.95 (dd, 2H), 5.10 (s, 4H), 5.10 (s, 4H), 5.20 (br d, 2H), 6.23 (d, 2H), 7.30-7.40 (m, 10H).Analysis calculated for C₄₀H₆₂N₄O₈: C, 66.12; H, 8.54; N, 7.71. Found: C, 66.04; H, 8.59; N, 7.70.

Example 85

A. (2S,3R,4R,5S)-1,6-Diphenyl-2,5-di-((N-toluenesulfonyl)amino)-3,4-O-isopropylidenehexanediol

To a stirred suspension of 411 mg (2.0 mmol) of CuBr-Me₂S in 2 mL of ether at 0 °C was added 3.08 ml (4.0 mmol) of 1.3 M phenyllithium in ether/cyclohexane. After 30 minutes, a suspension of 246 mg (0.50 mmol) of (2S,3R,4R,5S)-1,2:5,6-diimino-3,4-O-isopropylidene-hexanediol (703 mg, 1.43 mmol (Y.L. Merrer, et al, Heterocycles, 1987, 25, 541-548)) was added in 20 mL. After 80 minutes, the reaction mixture was quenched with 10% NH₄OH saturated with NH₄Cl. The ether layer was washed with brine, dried over magnesium sulfate, and concentrated under reduced pressure. Chromatography of the residue on silica gel with 7:3/hexane:EtOAc afforded 280 mg (86.4%) of the title compound. MS m/z 649 (M+H⁺)

B. N,N-Bis-(Cbz-valinyl)-(2S, 3R, 4R, 5S)-1,6-diphenyl-2,5-diamino-3,4-hexanediol

The resultant compound of Example 85A was converted to the title compound in a manner analogous to that described in Examples 84B and 84C

Example 86

N,N'-Bis-(1'-benzyloxycarbonyl-2'-methyl-1'-propyl)-3,4-dihydroxy-2,5-bis(phenylmethyl)adipamide.

A solution of 100 mg (0.68 mmol) of 2,3:4,5-dianhydro-D-iditol (R.S. Tipson, et al, Carbohydrate Research, 1968, 7, 232-243) in 5 mL of dichloromethane and 1 mL of DMSO was added to a solution of 208 mg (1.64 mmol) of oxalyl chloride in 10 mL of dichloromethane which had been treated with 257 mg (3.28 mmol) of DMSO at -78 °C. After 15 minutes at this temperature 685 mg (6.8 mmol) of triethylamine was added and the resulting solution allowed to warm to room temperature. The resulting dialdehyde is purifed by chromatography on silica gel and then oxidized to dimethyl 2,3:4,5-diepoxyadipate according to the procedure of D.R. Williams, et al (Tetrahederon Letters, 1988, 5087-5090). The diepoxide is then treated with the cuprate reagent prepared from benzyl lithium and CuBr-Me₂S or CuCN in an ethereal solvent such as tetrahydrofuran or diethyl ether, to affored dimethyl 3,4-dihydroxy-2,5-bis(phenylmethyl)adipate. The resulting diol is then protected as the bis(t-butyldiphenylsilyl) ether by treatment with t-butyldiphenylsilyl chloride in DMF in the presence of imidazole. Saponification of the diester with LiOH in aqueous THF, followed by acidification and coupling to valine benzyl ester in the manner described in Example 304, followed by deprotection with tetrabutylammmonium fluoride in THF affords the title compound.

Example 87

N,N-Bis-(Cbz-valinyl)-(2S, 3S, 4S, 5S)-1,6-diphenyl-2,5-diamino-3,4-hexanediol

Oxidation of of 2,3:4,5-dianhydro-D-iditol (R.S. Tipson, et al, Carbohydrate Research, 1968, 7, 232-243) as described in Example 86 but substituting THF for the dichloromethane/DMSO mixture as solvent, affords a solution of the corresponding dialdehyde. This solution is then recooled' to -78 °C, and treated with four equivalents (relative to the iditol) of phenylmagnesium bromide. The reaction mixture is then quenched with a pH 7 phosphate buffer and extracted into ethyl acetate, dried over sodium sulfate, and concentrated under reduced pressure. The resulting diol is treated with benzyl isocyanate, in the presence of either DMAP or diisopropylethylamine, in an inert solvent, such as benzene or THF. The resulting bis-carbamate is then treated with 2 equivalents of NaH or potassium t-butoxide in THF. The resulting diol is then treated with hydrogen over a Pd catalyst in a solvent such as MeOH to afford (2S, 3S, 4S, 5S)-3,4-dihydroxy-1,6-diphenyl-hexane-2,5-diamine. This is then converted into the title compound by treatment with Cbz-valine p-nitrophenyl ester in THF in the presence of triethylamine.

Example 88

2-(N-Benzyl-N-(benzyloxycarbonyl)amino)-5-(t-butyloxycarbonylamino)-1,6-diphenyl-3-hexene-3,4-oxide.

A solution of 160 mg (0.27 mmol) of the resultant compound of Example 16A and 117 mg (0.54 mmol) of 3-chloroperoxybenzoic acid in 1 ml of dichloromethane was stirred at ambient temperature for 2 days. The resulting solution was diluted with dichloromethane, washed sequentially with aqueous sodium bicarbonate and saturated brine, dried over MgSO₄, and concentrated Silica gel chromatography of the residue using 10% ethyl acetate in hexane provided 150 mg (92%) of the desired compound (R_f 0.50, 20% ethyl acetate in hexane) as an oil. Mass spectrum: (M + H)⁺ = 607.

Example 89

2-Amino-5-(t-butyloxycarbonylamino)-1,6-diphenyl-3-hexene-3,4-oxide.

A mixture of the resultant compound of Example 88 (152 mg) and 50 mg of 10% palladium on carbon in 50 ml of methanol was shaken under 4 atmospheres of hydrogen for 1 day. The mixture was filtered and concentrated in vacuo to provide 92 mg (96%) of the desired compound. Mass spectrum: (M + H)⁺ = 383.

Example 90

2,5-Di-(t-butyloxycarbonylamino)-1,6-diphenyl-3-hexene-3,4-oxide.

Using the procedure of Example 46 with the resultant compound of Example 89 provided the desired compound.

Example 91

(2S,3R,4R,5S)-2,5-Di-(N-(quinoline-2-carbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Quinaldic acid was coupled to the resultant compound of Example 28 using the carbodiimide coupling procedure of Example 8 to provide the desired compound.

Example 92

(2S,3S,4S,5S)-2,5-Di-(N-(valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 13C with the resultant compound of Example 49 provided the desired compound.

Example 93

(2S,3S,4S,5S)-2,5-Di-(N-(quinoline-2-carbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Quinaldic acid was coupled to the resultant compound of Example 92 using the carbodiimide coupling procedure of Example 8 to provide the desired compound.

Example 94

(2S,3S,4R,5S)-2,5-Di-(N-(valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.

Using the procedure of Example 13C with the resultant compound of Example 48 provided the desired compound.

Example 95

(2S,3S,4R,5S)-2,5-Di-(N-(quinoline-2-carbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Quinaldic acid was coupled to the resultant compound of Example 94 using the carbodiimide coupling procedure of Example 8 to provide the desired compound.

Example 96

(2S,3R,4R,5S)-2,5-Di-(N-(indole-2-carbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Indole-2-carboxylic acid was coupled to the resultant compound of Example 28 using the carbodiimide coupling procedure of Example 8 to provide the desired compound.

Example 97

(2S,3S,4S,5S)-2,5-Di-(N-(indole-2-carbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Indole-2-carboxylic acid was coupled to the resultant compound of Example 92 using the carbodiimide coupling procedure of Example 8 to provide the desired compound.

Example 98

(2S,3R,4S,5S)-2,5-Di-(N-(indole-2-carbonyl)-valinyl-amino)-3,4-dihydroxy-1,6-diphenylhexane.

Indole-2-carboxylic acid was coupled to the resultant compound of Example 94 using the carbodiimide coupling procedure of Example 8 to provide the desired compound.

Example 99

(2S,3R,4R,5S,2'S,2"S)-2,5-Di-(N-(2-(1,2,3,4-tetrahydropyrrolo[3,4-b]indol-3-on-2-yl)-4-methylpentanoyl)-amino)-3,4-dihydroxy-1,6-diphenylhexane.

(1'S)-2-(1-Carboxy-3-methylbutyl)-1,2,3,4-tetrahydropyrrolo[3,4-b]indol-3-one lithium salt, prepared according to the procedure of Kempf et. al. (J. Org. Chem. 1990, 55, 1390) was coupled to the resultant compound of Example 26 using the carbodiimide coupling procedure of Example 20D to provide the desired compound.

Example 100

(2S,3R,4R,5S,2'S,2"S)-2,5-Di-(N-(2-(1,2,3,4-tetrahydropyrrolo[3,4-b]indol-3-on-2-yl)-3-methylbutanoyl)-amino)-3,4-dihydroxy-1,6-diphenylhexane.

(1'S)-2-(1-Carboxy-2-methylpropyl)-1,2,3,4-tetrahydropyrrolo[3,4-b]indol-3-one lithium salt, prepared according to the procedure of Kempf et. al. (J. Org. Chem. 1990, 55, 1390) was coupled to the resultant compound of Example 26 using the carbodiimide coupling procedure of Example 20D to provide the desired compound.

Example 101

(2S,3R,4R,5S)-2,5-Di-(N-(((t-butyloxy)carbonyl)methyl)-amino)-1,6-diphenyl-3,4-dihydroxyhexane.

Using the procedure of Example 44A but replacing L-valine methyl ester hydrochloride with the resultant compound of Example 26 and replacing benzyl bromoacetate with t-butylbromoacetate provided the desired compound.

Fluorogenic Assay for Screening Inhibitors of HIV Protease

The inhibitory potency of the retroviral protease inhibiting Compounds disclosed herein can be determined by the following method.
Such a compound is dissolved in DMSO and a small aliquot further diluted with DMSO to 100 times the final concentration desired for testing. The reaction is carried out in a 6 X 50 mm tube in a total volume of 300 microliters. The final concentrations of the components in the reaction buffer are: 125 mM sodium acetate, 1 M sodium chloride, 5 mM dithiothreitol, 0:5 mg/ml bovine serum albumin, 1.3 uM fluorogenic substrate, 2% (v/v) dimethylsulfoxide, pH 4.5. After addition of inhibitor, the reaction mixture is placed in the fluorometer cell holder and incubated at 30°C for several minutes. The reaction is initiated by the addition of a small aliquot of cold HIV protease. The fluorescence intensity (excitation 340 nM, emmision 490 nM) is recorded as a function of time. The reaction rate is determined for the first six to eight minutes. The observed rate is directly proportional to the moles of substrate cleaved per unit time. The percent inhibition is 100 X (1 - (rate in presence of inhibitor)/(rate in absence of inhibitor)).
Fluorogenic substrate: Dabcyl-Ser-Gln-Asp-Tyr-Pro-Ile-Val-Gln-EDANS wherein DABCYL = 4-(4-dimethylaminophenyl)azobenzoic acid and EDANS = 5-((2-aminoethyl)amino)-naphthalene-1-sulfonic acid.

Retroviral protease inhibiting compounds disclosed herein inhibit HIV-1 protease at concentrations between 0.01 nM and 500,000 nM. Table 3 shows the inhibitory potencies of specific compounds against HIV-1 protease.

Compound of Example	Percent Inhibition	Inhibitor Concentration (micromolar)
10	100	270
25B	40	0.01
27B	74	0.001
31	58	0.005
47	44	0.01
48	56	0.001
49	65	0.001
50	67	0.1
52	86	0.005
53	73	0.005
54	81	0.001
55	84	0.001
56	76	0.001
57	45	0.001
58	68	0.001
59	62	0.001
60	78	0.1
61	38	0.001
62	62	0.005
63	74	0.005
66	44	0.002
67	78	0.001
68	69	0.001
69	67	0.005
71	68	0.01
73	45	0.01
75	69	0.001
76	74	0.001
77	33	0.01
78	40	0.001
79	60	0.1
80	56	0.05

Antiviral Activity

The anti-HIV activity of compounds disclosed herein can be determined by the following method.
A mixture of 0.1 ml (2 X 10⁵ cells/ml) of H9 cells and 0.1 ml (100 infectious units) of HIV-1_3B was incubated on a shaker for 2 h at 37°C. The resulting culture was washed three times and resuspended into 2 ml of medium containing 10 ul of a compound of the invention in dimethylsulfoxide. The control culture was treated in an identical manner except no compound was added to the medium. Aliquots of culture supernatants were removed at 3 time points, usually 4, 7 and 10 days, and monitored for HIV-1 antigen EIA (HIVAG-1) (Paul, et al., J. Med. Virol., 22 357 (1987)). Cell viability was determined by trypan blue dye exclusion, and cells were refed with media containing compound (except for control wells which were refed with media only) at these time points. Per cent inhibition of HIV by the compound was determined by comparing HIV antigen levels in the supernatants of infected cells incubated with compound to supernatants from the control culture without compound. The IC₅₀ is the concentration of compound that gives 50% inhibition of HIV activity. The LD₅₀ is the concentration of compound at which 50% of the cells remain viable.

Table 4 shows the inhibitory potencies of compounds disclosed herein against HIV-1 in H9 cells:

Compound of Example	IC₅₀ (micromolar)	LD₅₀ (micromolar)
27	0.12	11
47	2.3-4.5	25
48	0.015-0.027	60
49	0.05-0.07	>100
52	0.54	30
54	0.10	>100
55	0.04-0.1	>100
57	0.42	>100
58	0.05-0.1	>100
59	0.09-0.18	>100
63	0.27	>100
75	0.4-1.6	>100
76	0.25	180
77	1.2-7.5	>100
78	0.3-0.9	45

Claims

A compound of the formula:
or a hydrochloride salt thereof or an N-protected derivative thereof.
A compound according to Claim 1 wherein at each occurrence the N-protecting group is independently selected from the group consisting of acyl, acetyl, pivaloyl, t-butylacetyl, t-butyloxycarbonyl, benzyloxycarbonyl, benzoyl, or an L- or D-aminoacyl residue N-protected by acyl, acetyl, pivaloyl, t-butylacetyl, t-butyloxycarbonyl, benzyloxycarbonyl or benzoyl.
A compound selected from the group consisting of:

2-(N-benzylamino)-5-(t-butyloxycarbonylamino)-3,4-dihydroxy-1,6-diphenylhexane;

2-(N-benzyl-N-(benzyloxycarbonyl)amino)-5-(t-butyloxycarbonylamino)-3,4-dihydroxy-1,6-diphenylhexane;

5-amino-2-(N-benzylamino)-3,4-dihydroxy-1,6-diphenylhexane dihydrochloride;

2-amino-5-(t-butyloxycarbonylamino)-3,4-dihydroxy-1,6-diphenylhexane;

2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;

(2S,3R,4R,5S)-2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;

(2S,3S,4S,5S)-2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;

(2S,3R,4S,5S)-2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;

(2S,3R,4R,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane or a hydrochloride salt thereof;

(2S,3S,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane or a hydrochloride salt thereof;

(2S,3R,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane or a hydrochloride salt thereof;

(2S,3S,4R,5S)-5-amino-2-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenythexane;
and

(2S,3R,4S,5S)-2,5-di-(N-((isopropylaminocarbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.
A compound selected from the group consisting of:

(2S,3R,4R,5S)-2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;

(2S,3S,4S,5S)-2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane; and

(2S,3R,4S,5S)-2,5-di-(N-((t-butyloxy)carbonyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.
A compound selected from the group consisting of:

(2S,3R,4R,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane;

(2S,3S,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane;
and

(2S,3R,4S,5S)-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane;

or a hydrochloride salt thereof.
A compound selected from the group consisting of:

(2S,3R,4S,5S)-2,5-di-(N-(Cbz-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;

(2S,3S,4S,5S)-2,5-di-(N-(Cbz-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;

(2S,3R,4R,5S)-2,5-di-(N-(Cbz-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;

(2S,3S,4S,5S)-2,5-di-(N-(valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;

(2S,3R,4S,5S)-2,5-di-(N-(valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane;

(2S,3R,4R,5S)-2,5-di-(N-(valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane; and

(2S,3S,4R,5S)-2-(N-(t-butyloxy)carbonyl)amino)-5-(N-(Cbz-valinyl)amino)-3,4-dihydroxy-1,6-diphenylhexane.
A compound selected from the group consisting of:

2-(N-benzyl-N-(benzyloxycarbonyl)amino)-5-(t-butyloxycarbonylamino)-1,6-diphenyl-3-hexene-3,4-oxide;

2-amino-5-(t-butyloxycarbonylamino)-1,6-diphenyl-3-hexene-3,4-oxide; and

2,5-di-(t-buty(oxycarbonylamino)-1,6-diphenyl-3-hexene-3,4-oxide.